lysing enzymes. Enzyme preparations
Introduction to work
The state of the issue and the urgency of the problem
Lytic enzymes* that destroy bacterial cell walls were first discovered in human saliva and described by Alexander Fleming in 1922 (Fleming, 1922). The substance was named lysozyme, which means "an enzyme that dissolves bacteria." In 1929, Fleming first described the antibacterial properties of the fungus. Penicillum notatum - producer of the first industrial antibiotic penicillin, for which in 1945, in collaboration with Ernest Cheyne and Howard Flory, he was awarded the Nobel Prize. After the release of penicillin and until now, constant work has been carried out to create and produce new antibiotics, which is caused not only by the need to obtain substances of the required specificity and better quality, but also by the constant emergence of pathogenic microbes that are resistant to any, even the newest antibiotic (). The resulting unfavorable situation in the treatment of infectious diseases forces us to look for new effective antimicrobial agents. Many researchers point to the prospects of using lytic enzymes for this purpose, since the way they act on microbes, namely, the dissolution of the microbial cell, allows us to hope that pathogens resistant to them will not appear.
* The term "lytic enzymes" now refers to hydrolases that destroy the structural polymers of the cell walls of various microorganisms. Depending on which microorganisms they destroy, lytic enzymes are divided into bacteriolytic, yeast, mycolytic. By substrate specificity, they can be subdivided into chitinases, proteases, peptidoglycan hydrolases, and glucanases. It depends on which polymers, which are part of the cell membranes of various microorganisms, destroy lytic enzymes. In turn, for example, peptidoglycan hydrolases that destroy peptidoglycan, a structural component of bacterial cell walls, depending on which bond in the peptidoglycan molecule they hydrolyze, are divided into glycosidases (N-acetylglucosaminidases and N-acetylmuramidases (lysozymes)), amidases and endopeptidases. Of particular note are bacteriolytic proteases. Among the large number of currently known bacteriolytic proteases, there are very few. But it is these enzymes in the lytic group that have the widest spectrum of action against microorganisms. They are able to destroy the cell membranes of bacteria, yeast, filamentous fungi, and protozoa.
Depending on the context, the terms "lytic enzymes", "bacteriolytic enzymes", "peptidoglycan hydrolases", "yeast enzymes", "lytic proteases" will be used in this work.
In the period from the 5th to the 70s of the 20th century, intensive work was carried out to find the producers of such enzymes, to isolate them, and to study their properties. It is now known that many living organisms - from viruses to humans - produce lytic enzymes. Among the bacteria, enzyme producers were found that actively lyse not only cells of competing bacteria, but also cells of microorganisms of other systematic groups - yeast, filamentous fungi, and protozoa. For such bacteria, an order was formed in 1978 Lysobacteria, including family Lysobacteraceae and genus Lysobacter combining four species (Christensen and Cook, 1978). Bacteria that previously belonged to other genera, but differed from their typical representatives in a number of properties, and most importantly in their political ability, were transferred to this systematic group. Subsequently, interest in lysing bacteria somewhat weakened, but now they have again become of interest to researchers. During the first decade of the 21st century, eleven new species of the genus Lysobacter. As a result of ongoing work on the systematization of known microorganisms, the systematic position of the genus was also corrected. Lysobacter. It is now included in the family Xanthomonadaceae(Bergey's Manual of Systematic Bacteriology, 2001). In the literature, one can still observe obvious confusion in the systematic position of the described producers of lytic enzymes. For example, a producer of enzymes that are similar in all properties to enzymes of the type species of the genus Lysobacter - Lysobacter enzymogenes - referred to by the authors as Achromobacter lyticus(Shiraki et ah, 2002, Life or/., 1997).
For bacteria producing extracellular lytic enzymes, as for
any bacteria, intracellular autolytic enzymes are vital, which destroy covalent bonds in peptidoglycan, the main structural component of their cell wall, and thus play a major role in the processes of growth and division. In the cells of bacteria producing extracellular lytic enzymes, there is a parallel synthesis and movement through the cytoplasmic membrane to the site of their action of both autolytic enzymes, which can be localized in the membrane, periplasm and cell wall, and extracellular bacteriolytic enzymes secreted into the environment. In this regard, it is logical to ask about the mechanism and regulation of the process of the simultaneous functioning of these enzymes, whether autolytic enzymes can be precursors of extracellular bacteriolytic enzymes? To date, a large number of works have been published on the isolation and characterization of both extracellular and intracellular bacteriolytic enzymes of bacteria. However, so far
So far, there is no information on a comparative study of extra- and intracellular lytic systems in the same bacterium. Autolytic enzymes have been well studied in many representatives of gram-positive bacteria (Shockman and Holtje, 1994), in gram-negative ones, with the exception of Escherichia coli(Holtje and Tuomanen, 1991), they have not been studied in detail.
In 1973, at the IBFM of the USSR Academy of Sciences (IBPM RAS), by order of the Academy of Sciences, work began on the topic “Creation of effective means of combating pathogenic microorganisms resistant to antibiotics”. The cultural fluid of a gram-negative bacterium isolated in 1976 from the water of the Oka River in the area of the wastewater treatment plant in Pushchino, Moscow Region, was the basis of a drug called lysoamidase and possessing bacteriolytic and proteolytic activities. Successful clinical trials of lysoamidase allowed it to be registered as a drug for the treatment of external infections caused by gram-positive microflora. According to a number of morphological and biochemical features, the producing bacterium was presumably assigned to the genus Xanthomonas. However, according to a number of essential properties, for example, the lack of mobility, the lysoamidase producer differed from bacteria of this genus.
Purpose and tasks of the work
The purpose of the work is to study the biochemical and genetic features of the functioning and the relationship between the intracellular and extracellular lytic systems of the bacterium-producer of the lysoamidase preparation in order to create a new generation of antimicrobial drugs based on the information obtained.
Main goals:
clarification of the taxonomic position of the bacterium-producer;
determination of the structure of the peptidoglycan of the bacterium-producer - the substrate of autolytic enzymes;
isolation and characterization of extracellular lytic enzymes of the producing bacterium;
isolation and characterization of intracellular (autolytic) enzymes of the producer;
determination of the structure of genes of extracellular lytic enzymes
producer;
study of the features of the interaction of lytic enzymes with
various target microorganisms;
production of recombinant lytic enzymes of the producer and study
their properties to assess the possibility of using such enzymes as
fundamentals of new antimicrobials;
study of the possibility of using lysoamidase and various forms of lytic enzymes of the producer for the treatment of "internal" infections on the example of anthrax.
Scientific novelty of the work
Based on the established morphological, biochemical and genetic properties, the bacterium producing the antimicrobial drug lysoamidase is assigned to the genus Lysobacter. The strain studied in this work Lysobacter sp. XL1 was obtained by selection from the original culture and deposited in the All-Russian Collection of Microorganisms (VKM V-2249D). It has been established that during long-term cultivation of this lytically highly active strain on media that promote the secretion of extracellular products, cells of a lytically low-active strain arise and accumulate in the population due to a higher growth rate. Lysobacter sp. XL2.
The endocellular and exocellular lytic systems of the same bacterium were characterized for the first time using the example Lysobacter sp. XL1 and XL2. The endocellular system of both strains contained nine enzymes of different substrate specificity and localization (glucosidases, amidases, endopeptidases). As part of the extracellular lytic system Lysobacter sp. XL1 found five enzymes, including muramidase (LZ), amidase (L2), three endopeptidases (L1, L4, L5). Extracellular lytic system of a low-activity strain Lysobacter sp. XL2 consists of muramidase and amidase. The properties of enzymes of different lytic systems differ significantly from each other: intracellular enzymes are acidic proteins that are active at 29C - the temperature of optimal bacterial growth, high ionic strength of the medium and alkaline pH; extracellular enzymes are alkaline proteins that are active at low ionic strengths, alkaline pH and high temperatures (50-80C).
For the first time, it was revealed that the post-secretory electrostatic interaction of a high-molecular acid polysaccharide and enzymes Lysobacter sp. XL1 leads not only to a significant stabilization of enzymes, but also, in some cases, to a change in their activity. The polysaccharide enhances the action of muramidase on the cells of Staphylococcus aureus, and the lytic enzymes associated with the polysaccharide become capable of destroying resting spores of bacteria of the genus bacillus. Polysaccharide Lysobacter sp. XL1 completely inhibits the activity of a number of lytic enzymes of other producers. Obviously, the formation of such extracellular complexes by microorganisms is ecologically significant for them.
It was shown for the first time that extracellular lytic enzymes L2 and L5 Lysobacter sp. enter the space surrounding the cell inside the outer membrane vesicles formed by the bacterium. Enzymes enclosed in vesicles are able to lyse living cells of representatives of various groups of microorganisms, for example, gram-negative bacteria of the genera Pseudomonas, Proteus, Erwinia, Alcaligenes, Gram-positive bacteria belonging to the genera Bacillus, Micrococcus, Staphylococcus, Rothayibacter, yeast of the genus Candida filamentous fungus Sclerotinia sclerotiorum, in contrast to lytic enzymes outside the vesicles. Thus, such a pathway of secretion of lytic enzymes is of great biological importance for the producer cell, since it expands the spectrum of microorganisms with which it can compete in nature.
Important features of the interaction of extracellular lytic enzymes have been established Lysobacter sp. with native target cells. For effective hydrolysis of gram-positive bacteria cells, enzymes require preliminary contact with a negatively charged cell wall polymer (teichoic or teichuronic acids), while the chemical structure of the polymer is not critical. Native cells of gram-negative bacteria lytic enzymes Lysobacter sp. (3a with the exception of L5) are destroyed only if the outer membrane of the target cell is previously destabilized by a suitable method (temperature, polymyxin B, gentamicin, amikacin). Lytic enzyme L5 destroys cells of gram-negative bacteria without pretreatment.
The practical significance of the work
On the basis of the data obtained, a new procedure for obtaining the lysoamidase preparation with a high yield of the target product (up to 80%) was developed and scaled up.
Methods for obtaining two recombinant lytic endopeptidases have been developed Lysobacter sp. XLl using heterologous systems based on E. coli(refolding from inclusion bodies) and Pseudomonas fluorescens(purification of secreted proteins).
The possibility of using the drug lysoamidase, as well as vesicles Lysobacter sp. XLl for the treatment of various forms of experimental anthrax.
Based on the dissertation materials, patents of the Russian Federation were obtained: No. 2139348 (1999), No. 2193063 (2002), No. 2296576 (2007), No. 2407782 (2010), No. 2408725 (2011), USA patent No. 7,150,985 B2 (2006), China patent No. 274608 (2006), European Patent No. 1902719 B1 (2011).
The new data obtained in the work are used in biochemistry courses at the Faculty of Biology of Moscow State University named after M.V. Lomonosov and biological faculties of other higher educational institutions.
Approbation of work
The dissertation materials were presented at the third and fourth All-Union conference "Biosynthesis of enzymes by microorganisms", Kobuleti, 1986; Tashkent, 1988; Second All-Union Conference "Wounds and wound infection", Moscow, 1986; 14 International Congress of Biochemistry, Prague, CSSR, 1988; All-Union Conference "Regulation of microbial metabolism", Pushchino, 1989; Fifth International Conference on Chemistry and Biotechnology of Active Natural Compounds, Varna, Bulgaria, 1989; 1 International symposium "Molecular organization of biological structures", Moscow, 1989; 5 European congress on Biotechnology, Copenhagen, 1990; International conference on antimicrobial activity of nonantibiotics, Copenhagen, 1990; Conference “Biosynthesis and degradation of microbial polymers. Fundamental and Applied Aspects”, Pushchino, 1995; International Conference "Microbial polysaccharide", Canada, 1995; First International Conference "Polysaccharide Engineering" Trondheim, Norway, 1995; Conferences of surgeons, Kaluga, 1996; IV symposium "Chemistry of proteolytic enzymes", Moscow, 1997; Second Congress of the Biochemical Society of the Russian Academy of Sciences, Moscow, 1997;
seminar-presentation of innovative scientific and technical projects
"Biotechnologies of the Moscow Region-97", Pushchino, 1997; International symposium "Modern problems of microbial biochemistry and biotechnology", Pushchino, 2000; international conference "Biotechnology at the turn of two millennia", Saransk, 2001; 3 Congress of the Biochemical Society, St. Petersburg, 2002; All-Russian Conference “Problems of Medical Enzymology. Modern technologies of laboratory diagnostics of the new century”, Moscow, 2002; Second, Fifth, Sixth Moscow International Congresses "Biotechnology: state and development prospects", Moscow, 2003, 2009, 2011; First All-Russian Congress "Advances in Medical Mycology", Moscow, 2003; III Conference "Biotechnology: State and Perspective of Investigation", Moscow, 2005; Third All-Russian school-conference "Chemistry and biochemistry of carbohydrates", Saratov, 2004; Conferences "Fundamental sciences-medicine", Moscow, 2005; conference "Results of fundamental and applied research for the creation of new drugs", Moscow, 2008; All-Russian Conference "Ecotoxicology-2010", Tula, 2010; Conference "Fundamental Sciences for Medicine", Moscow, 2010, All-Russian Symposium with international participation "Biologically active substances of microorganisms: past, present, future", Moscow, 2011.
Publications
Structure and scope of work
Federal Agency for Education
State educational institution
higher professional education
Perm State Technical University
Department of Chemistry and Biotechnology
Abstract:
lytic enzymes. Lysozyme
Performed:
student gr.KhTBmPiB-05
Shevchenko I.K.
Supervisor:
Ph.D. Gryaznova D.V.
Perm, 2010
Lytic enzymes of microbial origin 5
Discovery history 5
Localization and physiological role of bacteriolytic enzymes in bacterial cultures 6
Influence of the structure of bacterial cell walls on the lytic ability of enzymes 7
Peptidoglycan of bacterial cell walls. eight
Substrate specificity of bacteriolytic enzymes 9
Discovery of the bacteriolytic complex "Lysoamidase" 10
Prospects for the use of lysoamidase in medicine 10
LYSOZYME 12
Lysis mechanism 13
Conclusion. fifteen
Literature. 16
Introduction.
The problem of lysis of the cell walls of microorganisms of various taxonomic groups in order to increase their nutritional value and obtain biologically active substances contained in the protoplasm of microbial cells in an undegraded form is relevant and of national economic importance.Various physical and biochemical methods are known for destroying microbial biomass. The enzymatic method of destruction of cell walls, in contrast to physicochemical methods, allows for a controlled effect on cells and the extraction of target products from them.
Among the enzymes produced by microorganisms, a special place is occupied by lytic enzymes that catalyze biochemical reactions of sequential degradation of the structural elements of the microbial cell wall.
The use of lytic enzyme preparations makes it possible to intensify the release of many valuable physiologically active substances from the microbial biomass: enzymes, vitamins, amino acids, etc. It is known that the cell wall of fodder yeast prevents the absorption of cytoplasmic substances of the cell when fed to animals. The biomass of fodder yeast after enzymatic lysis of cell walls has an increased nutritional value, which makes it possible to use it more efficiently in feed production, including as part of whole milk substitutes for young farm animals.
Also, lytic enzymes can be used in environmental protection technologies, at the stages of utilization of microbial biomass, which is a waste of microbiological production.
In addition, antimicrobial drugs based on lytic enzymes have recently been developed, including those for the treatment of dermatomycosis, which have a number of advantages over chemically synthesized fungicides. Positive results have been obtained when using them for the treatment of diseases of the gastrointestinal tract of farm animals. The use of lytic enzymes is promising in the fight against staphylococcal infection, in the treatment of dental caries.
Of particular interest for the creation of industrial production of enzymes are thermotolerant microorganisms, including actinomycetes-producers of lytic enzymes, which have a high growth rate and resistance to changes in cultivation temperature. It is also important that thermotolerant cultures are often more competitive than mesophilic producers in terms of infecting microflora.
Lytic enzymes of microbial origin
It is well known that cells of bacteria, fungi, and higher plants, in contrast to animal cells, have, as a rule, very strong cell walls. This is due to the need for these organisms to withstand the numerous biological, chemical and physical factors of their habitat. At the same time, to carry out many experiments in the field of modern cellular and molecular biology, it is necessary to have "bare" cells of these organisms, devoid of thick cell walls. Such "naked" cells, or "protoplasts", are widely used for experiments on cell fusion, for various genetic engineering manipulations, etc. In connection with these problems, the close attention of scientists has long been attracted by specific enzymes (biological catalysts of a protein nature) capable of destroying (lysing) the cell walls of bacteria, fungi, and higher plants. By the way, the progress achieved so far in studying the structure and functioning of the surface structures of such cells is largely associated with the development of work on the enzymatic lysis of the cell walls of these organisms.It turned out that cell wall-destroying (lytic) enzymes are found in significant amounts in these structures themselves, in the immediate vicinity of the objects of their action. Such enzymes are called endogenous (intracellular). In addition, it has been established that some of the lytic enzymes are exogenous, that is, they are secreted (released) into the habitat of the organisms that form them.
Localization and physiological role of bacteriolytic enzymes in bacterial cultures
If we talk about the location of bacteriolytic enzymes in a bacterial culture, then we should first of all divide them into three groups in this regard.
The first group consists autolysins - bacteriolytic enzymes that are always present (in an active or inactive state) in the cell wall itself. They take part in the process of growth and differentiation of bacterial cells. In a bacterial cell, apparently, in the norm there is a relationship between the activities of enzymes that destroy and synthesize components of the cell wall. Indeed, the incorporation of newly synthesized materials into the cell wall cannot occur without prior cleavage of certain chemical bonds.
The processes of lysis and biosynthesis of the wall occur simultaneously with the growth and development of the bacterial cell, and only at the later stages of development, when biosynthetic processes subside and the activity of lytic enzymes remains at the same level, bacterial cell lysis occurs.
The second group includes lytic enzymes of bacterial spores . They are activated along with other enzymes involved in the degradation of biopolymers during the period of sporulation (spore formation) and during the germination of bacterial spores. These enzymes are involved in the processes of destruction of the shell and as autolysins in the processes of growth and morphogenesis of a bacterial cell.
Finally, the third group is extracellular lytic enzymes . Their biological role seems to lie in the fact that bacteria synthesizing and secreting such enzymes into the environment have an advantage over other bacteria, primarily in food sources. Destroying the cells of other bacteria, the bacterium producing lytic enzymes uses amino acids, carbohydrates and other components of the lysed cell for its own needs. In addition, this group of bacteriolytic enzymes plays an unquestionably important role in protecting the cells that secrete these enzymes into the environment from other bacteria living in the same ecological niche.
Influence of the structure of bacterial cell walls on the lytic ability of enzymes
All bacteria can be divided into two significantly different groups: gram-negative and gram-positive. The difference is due to the fundamentally different structure of the cell walls of Gram-positive and Gram-negative bacteria. In gram-positive bacteria (eg, staphylococci or micrococci), the cell wall consists of a 70–80 nm thick multilayer structure called peptidoglycan. The peptidoglycan sac that covers the cytoplasmic membrane of these cells consists of polysaccharide chains interconnected into a single network by peptide bridges. It accounts for up to 80% of the weight of their cell membranes in Gram-positive bacteria. In addition to peptidoglycan, the cell walls of these bacteria include negatively charged polymers - the so-called teichoic acids (from the Greek "teichos" - wall). Some of the teichoic acids are covalently bound to the peptidoglycan network, and some to the lipids of the cytoplasmic membrane. In the latter case, they are called lipidteichoic acids. Teichoic acids, due to the presence of phosphoric acid in their composition, provide an electronegative charge on the surface of Gram-positive bacteria cells.In some Gram-positive bacteria (for example, Staphylococcus aureus), teichoic acids consist of several tens of ribitol phosphate molecules - ribitolteichoic acids, in other Gram-positive bacteria, these biopolymers consist of glycerol phosphate molecules (glycerolteichoic acids). Teichoic acids are present only in Gram-positive bacteria.
The main difference in the structure of the shells of gram-positive and gram-negative bacteria is the presence in the latter, in addition to the cytoplasmic one, of the so-called outer membrane. This structure, located above a thin, one-three-layer peptidoglycan sac (8 nm), is a typical two-layer membrane, in which quite a lot of fairly unique components have been identified: lipopolysaccharides, lipoproteins, and proteins - porins, from which pores are formed in the outer membrane, allowing to penetrate into the shell (and from it into the environment) relatively low molecular weight compounds.
Bacteriolytic enzymes cannot hydrolyze the peptidoglycan layer in whole cells of gram-negative bacteria without removing the outer membrane, which can only be achieved by treating these cells with chelating agents, detergents, or physical methods.
Peptidoglycan of bacterial cell walls.
It is primarily responsible for maintaining the shape of bacterial cells and is the structure that bacteriolytic enzymes act to destroy. It is important to emphasize here that peptidoglycan is necessarily present in all true bacteria, but its availability for the action of bacteriolytic enzymes in Gram-positive and Gram-negative bacteria differs significantly.As mentioned above, peptidoglycan consists of polysaccharide chains and peptide bridges that unite the entire structure into a single "bag" that surrounds the bacterial cell from the outside. Polysaccharide (glycan) chains are formed by the alternation of two "bricks" - nitrogen-containing derivatives of glucose: N-acetylglucosamine and N-acetylmuramic acid - and generally represent a chitin-like structure. This is interesting from an evolutionary point of view, since chitin and chitin-like structures are widespread in almost all representatives of the living world (excluding only plants) and are one of the most common biopolymers on Earth.
The structure of peptidoglycan glycan chains is the same in most studied bacteria. The peptide part of peptidoglycan in different bacteria can differ significantly. However, in all cases it is formed from 4-5 residues of L- or D-amino acids. These short peptides, on the one hand, are connected by their free NH2 group by an amide bond to the carboxyl of muramic acid, and on the other hand, they are linked to the same short peptide of the adjacent glycan chain. In Gram-positive bacteria, in particular Staphylococcus aureus, peptides associated with glycan chains do not bind directly to each other, but with the participation of an additional peptide, the so-called cross-linking bridge. In Staphylococcus aureus, this peptide bridge consists of five molecules of the simplest amino acid glycine. The presence of such bridges in the peptidoglycan structure of Gram-positive bacteria makes it seem to be more dense, which is one of the most important reasons for the retention of the dye by these cells when stained by Gram. Hydrolysis (cleavage with water) of certain bonds in peptidoglycan leads to degradation of the cell wall and lysis of bacteria.
Substrate specificity of bacteriolytic enzymes
According to substrate specificity, bacteriolytic enzymes are divided into three types.
The first type consists of the so-called glycosidases that destroy polysaccharide (glycan) chains. These include N-acetylmuramidase (lysozyme), which hydrolyses the bond between N-acetylmuramic acid and N-acetylglucosamine, and N-glucosaminidase, which hydrolyzes the bond between N-acetylglucosamine and N-acetylmuramic acid.
The second type is represented by one enzyme - N-acetylmuramyl-L-alanylamidase (or simply amidase), which cleaves the bond between the muramic acid of the polysaccharide and the peptide part.
The third type includes peptidases that hydrolyze the peptide bonds of peptidoglycan.
Discovery of the bacteriolytic complex "Lysoamidase"
In 1975, an interesting observation was made at the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences in Pushchino (on the banks of the Oka River). In the waters of the Oka below Pushchin, microbiologists G.K. Scriabin, V.A. Lambina et al. found a fairly extensive "sterile spot" that was practically free of bacteria. From water samples in the immediate vicinity of the "spot", a culture of bacteria of the genus Xanthomonas was isolated, which released into the environment a certain factor that inhibits the growth of many bacteria, including pathogenic ones. The biochemists of the Institute, under my leadership, established that the active antibacterial principle of this "factor" is a complex of high-molecular polysaccharide, charged electronegatively, and positively charged enzymes. The purified preparation of this complex was named lysoamidase. Already at the first stage of its biochemical study, it was found that it contains bacteriolytic enzymes capable of cleaving peptide (or amide) bonds in peptidoglycan, ultimately leading to the lysis of bacterial cells.Prospects for the use of lysoamidase in medicine
Already at the first stage of studying the properties of the lysoamidase preparation, it became clear that it can be successfully used not only in biology, for example, to obtain bacterial protoplasts devoid of cell walls (Fig. 4), but also in medicine. It turned out that the drug lysoamidase is an effective means of combating multiple antibiotic-resistant pathogenic microorganisms.Currently, one of the most important problems in medicine is the very rapid emergence in clinical forms of pathogenic bacteria of resistance (immunity) to antibiotics used in medical practice. For example, in most maternity hospitals both in Russia and in other countries, for the above reasons, it is becoming increasingly difficult to fight purulent infections caused, in particular, by bacteria such as staphylococci and streptococci. However, the drug lysoamidase has been shown to be very effective in lysing multidrug-resistant strains of staphylococci and other Gram-positive pathogenic bacteria.
Lysoamidase effectively kills clinical strains, which, at any concentration, are not affected by almost all antibiotics used in Russian clinics. Further biomedical and clinical trials of this drug led physicians to the conclusion that lysoamidase is an excellent means of combating purulent infections. It can be widely used in purulent surgery, dentistry, gynecology in the treatment of difficult-to-heal trophic ulcers, etc. Currently, the drug is approved for use in medical practice and its production has been established at the Vyshnevolotsk plant of enzyme preparations for medical purposes.
In biomedical and clinical testing of the drug, it turned out that it not only has a lytic effect on pathogenic bacteria, but also cleans wounds well from necrotic (dead) tissues, and also stimulates wound healing, having a powerful immunostimulating effect.
It turned out that the effective cleaning of wounds from necrotic masses (primarily consisting of denatured proteins) is associated with the presence in the lysoamidase preparation of not only bacteriolytic enzymes, but also proteases (protein-degrading enzymes). And the immunostimulating activity of lysoamidase is due to the presence of a polysaccharide in the preparation. The presence of a polysaccharide is of fundamental importance for the possibility of using lysoamidase in medicine, since the bacteriolytic enzymes present in lysoamidase are electrostatically bound to the polysaccharide, which leads to their significant stabilization. After separation from the polysaccharide, the bacteriolytic enzymes of lysoamidase, like their other previously known analogues, lose their enzymatic activity after a few days. As part of lysoamidase, these enzymes retain their activity in the cold almost unchanged for several years, which is a mandatory requirement for medical preparations.
LYSOCYME
Lysozyme (muramidase) is an antibacterial agent, an enzyme of the hydrolase class, which destroys the cell membranes of bacteria by hydrolyzing muramylglucosamine of the cell wall of gram-positive bacteria. Lysozyme is contained, first of all, in places where the animal body comes into contact with the environment - in the mucous membrane of the gastrointestinal tract, lacrimal fluid, breast milk, saliva, nasopharyngeal mucus, etc. Lysozyme is found in large quantities in saliva, which explains its antibacterial properties. In human breast milk, the concentration of lysozyme is very high (about 400 mg / l). This is much more than in the cow. At the same time, the concentration of lysozyme in breast milk does not decrease with time; six months after the birth of a child, it begins to increase.Discovered in 1922 by Alexander Fleming in mucus from the nasal cavity and then found in many tissues and fluids of the human body (cartilage, spleen, leukocytes, tears), in plants (cabbage, turnip, radish, horseradish), in some bacteria and phages and, in the largest amount, in egg white. Lysozyme from different sources differ in structure, but are similar in action. Egg white lysozyme is the first enzyme for which a three-dimensional structure was established by X-ray diffraction analysis and a relationship between the structure and mechanism of action was revealed (1965); these studies are a significant contribution to the understanding of the mechanisms of enzymatic catalysis in general.
Lysozyme is a protein with a molecular weight of about 14,000; the only polypeptide chain consists of 129 amino acid residues and is folded into a compact globule (30×30×45 Å). The three-dimensional conformation of the polypeptide chain is supported by 4 disulfide (- S - S -) bonds. (There are 3 of them in human milk lysozyme, 2 in goose egg white, they are not in T4 phage lysozyme; the more disulfide groups, the lysozyme is more stable, but less active.) Lysozyme globule consists of two parts, separated by a gap; in one part, most amino acids (leucine, isoleucine, tryptophan, etc.) contain hydrophobic groups; in others, amino acids (lysine, arginine, aspartic acid, etc.) with polar groups predominate. The polarity of the environment affects the ionization of two carboxyl groups (-COOH) located on the surface of the gap of the molecule from its different sides (see Fig.). Lysozyme acts on one of the main components of the bacterial wall - a complex polysaccharide consisting of two types of amino sugars. The polysaccharide is sorbed on the lysozyme molecule in the gap at the border of its hydrophobic and hydrophilic parts in such a way that 6 rings of amino sugars bind to the enzyme, and one of the glycosidic bonds connecting them (between 4 and 5 rings) is between carboxyls. Due to the interactions between the lysozyme carboxyls and the atoms forming the glycosidic bond, as well as the distortion of the valence angles of the substrate, the bond is activated and broken. This leads to the destruction of the bacterial cell membrane.
Rice. The structure of lysozyme. (N. A. Kravchenko)
Lysis mechanism
The enzyme attacks peptidoglycans (particularly murein) that make up the cell walls of bacteria (especially gram-positive ones). Lysozyme hydrolyses the (1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. In this case, peptidoglycan binds to the active site of the enzyme (in the form of a pocket) located between its two structural domains. The substrate molecule in the active site adopts a conformation close to that of the transition state. In accordance with the Phillips mechanism, lysozyme binds to a hexasaccharide, then converts the 4th residue in the chain into a twist chair conformation. In this stressed state, the glycosidic bond is easily broken.Residues of glutamic acid (Glu35) and aspartic acid (Asp52) are critical for enzyme function. Glu35 acts as a proton donor upon breaking the glycosidic bond of the substrate, destroying the bond, while Asp52 acts as a nucleophile during the formation of an intermediate, the glycosyl enzyme. Then the glycosyl enzyme reacts with a water molecule, as a result of which the enzyme returns to its original state and a hydrolysis product is formed.
Application
The drug lysozyme is used in the treatment of eyes, nasopharynx, gums, burns, in obstetrics, etc. It is registered in the food industry as a food additive E1105.
Conclusion.
These data indicate the important role played by bacteriolytic enzymes in the vital activity of bacteria and bacterial communities located in the same ecological niche.On the example of the drug lysoamidase and lysozyme, the prospects for the use of bacteriolytic enzymes as an effective therapeutic agent for combating pathogenic bacteria, including those that are multiple resistant to antibiotics, are demonstrated.
Despite the great interest shown in the problem of enzymatic lysis of microorganisms, there is no industrial production of lytic enzyme preparations in Russia. Therefore, the study of the conditions for the biosynthesis of lytic enzymes and the development of a technology for obtaining an enzyme preparation of lytic action for use in various sectors of the national economy and medicine is relevant and promising.
Literature.
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3. Zakharova I.Ya., Pavlova I.N. Lytic enzymes of microorganisms. Kyiv: Nauk. thought, 1985.
4. Skryabin G.K., Kulaev I.S. Lysoamidase - a challenge to microbes // Science in the USSR. 1990. No. 2. S. 52-53.
5. Kulaev I.S. Bacteriolytic enzymes of microbial origin in biology and medicine. // SOZH, 1997, No. 3, p. 23–31.
6. Phillips D., Three-dimensional structure of the enzyme molecule, in the collection: Molecules and cells, trans. from English, c. 3, M., 1968;
7. Wikipedia: free encyclopedia [Electronic resource]. – URL: http://ru.wikipedia.org/wiki/Lysozyme (date of access: 05/15/2010)
8. Academics: electronic encyclopedias [Electronic resource]. – URL: http://dic.academic.ru/dic.nsf/bse/103560/lysozyme (date of access: 05/15/2010)
Millions of chemical reactions take place in the cell of any living organism. Each of them is of great importance, so it is important to maintain the speed of biological processes at a high level. Almost every reaction is catalyzed by its own enzyme. What are enzymes? What is their role in the cell?
Enzymes. Definition
The term "enzyme" comes from the Latin fermentum - leaven. They may also be called enzymes, from the Greek en zyme, "in yeast."
Enzymes are biologically active substances, so any reaction that occurs in a cell cannot do without their participation. These substances act as catalysts. Accordingly, any enzyme has two main properties:
1) The enzyme speeds up the biochemical reaction, but is not consumed.
2) The value of the equilibrium constant does not change, but only accelerates the achievement of this value.
Enzymes speed up biochemical reactions by a thousand, and in some cases a million times. This means that in the absence of an enzymatic apparatus, all intracellular processes will practically stop, and the cell itself will die. Therefore, the role of enzymes as biologically active substances is great.
A variety of enzymes allows you to diversify the regulation of cell metabolism. In any cascade of reactions, many enzymes of various classes take part. Biological catalysts are highly selective due to the specific conformation of the molecule. Since enzymes in most cases are of a protein nature, they are in a tertiary or quaternary structure. This is again explained by the specificity of the molecule.
Functions of enzymes in the cell
The main task of the enzyme is to speed up the corresponding reaction. Any cascade of processes, from the decomposition of hydrogen peroxide to glycolysis, requires the presence of a biological catalyst.
The correct functioning of enzymes is achieved by high specificity for a particular substrate. This means that a catalyst can only speed up a certain reaction and no other, even a very similar one. According to the degree of specificity, the following groups of enzymes are distinguished:
1) Enzymes with absolute specificity, when only one single reaction is catalyzed. For example, collagenase breaks down collagen and maltase breaks down maltose.
2) Enzymes with relative specificity. This includes substances that can catalyze a certain class of reactions, such as hydrolytic cleavage.
The work of a biocatalyst begins from the moment of attachment of its active site to the substrate. In this case, one speaks of a complementary interaction like a lock and a key. Here we mean the complete coincidence of the shape of the active center with the substrate, which makes it possible to accelerate the reaction.
The next step is the reaction itself. Its speed increases due to the action of the enzymatic complex. In the end, we get an enzyme that is associated with the products of the reaction.
The final stage is the detachment of the reaction products from the enzyme, after which the active center again becomes free for the next work.
Schematically, the work of the enzyme at each stage can be written as follows:
1) S + E ——> SE
2) SE ——> SP
3) SP ——> S + P, where S is the substrate, E is the enzyme, and P is the product.
Enzyme classification
In the human body, you can find a huge number of enzymes. All knowledge about their functions and work was systematized, and as a result, a single classification appeared, thanks to which it is easy to determine what this or that catalyst is intended for. Here are the 6 main classes of enzymes, as well as examples of some of the subgroups.
- Oxidoreductases.
Enzymes of this class catalyze redox reactions. There are 17 subgroups in total. Oxidoreductases usually have a non-protein part, represented by a vitamin or heme.
Among oxidoreductases, the following subgroups are often found:
a) Dehydrogenases. The biochemistry of dehydrogenase enzymes consists in the elimination of hydrogen atoms and their transfer to another substrate. This subgroup is most often found in the reactions of respiration, photosynthesis. The composition of dehydrogenases necessarily contains a coenzyme in the form of NAD / NADP or flavoproteins FAD / FMN. Often there are metal ions. Examples are enzymes such as cytochrome reductase, pyruvate dehydrogenase, isocitrate dehydrogenase, and many liver enzymes (lactate dehydrogenase, glutamate dehydrogenase, etc.).
b) Oxidases. A number of enzymes catalyze the addition of oxygen to hydrogen, as a result of which the reaction products can be water or hydrogen peroxide (H 2 0, H 2 0 2). Examples of enzymes: cytochrome oxidase, tyrosinase.
c) Peroxidases and catalases are enzymes that catalyze the breakdown of H 2 O 2 into oxygen and water.
d) oxygenases. These biocatalysts accelerate the addition of oxygen to the substrate. Dopamine hydroxylase is one example of such enzymes.
2. Transferases.
The task of the enzymes of this group is to transfer radicals from the donor substance to the recipient substance.
a) methyltransferase. DNA methyltransferases, the main enzymes that control the process of nucleotide replication, play an important role in the regulation of the nucleic acid.
b) Acyltransferases. Enzymes of this subgroup transport the acyl group from one molecule to another. Examples of acyltransferases: lecithincholesterol acyltransferase (transfers a functional group from a fatty acid to cholesterol), lysophosphatidylcholine acyltransferase (an acyl group is transferred to lysophosphatidylcholine).
c) Aminotransferases - enzymes that are involved in the conversion of amino acids. Examples of enzymes: alanine aminotransferase, which catalyzes the synthesis of alanine from pyruvate and glutamate by amino group transfer.
d) Phosphotransferases. Enzymes of this subgroup catalyze the addition of a phosphate group. Another name for phosphotransferases, kinases, is much more common. Examples are enzymes such as hexokinases and aspartate kinases, which add phosphorus residues to hexoses (most often glucose) and to aspartic acid, respectively.
3. Hydrolases - a class of enzymes that catalyze the cleavage of bonds in a molecule, followed by the addition of water. Substances that belong to this group are the main digestive enzymes.
a) Esterases - break ester bonds. An example is lipases, which break down fats.
b) Glycosidases. The biochemistry of enzymes of this series consists in the destruction of glycosidic bonds of polymers (polysaccharides and oligosaccharides). Examples: amylase, sucrase, maltase.
c) Peptidases are enzymes that catalyze the breakdown of proteins into amino acids. Peptidases include enzymes such as pepsins, trypsin, chymotrypsin, carboxypeptidase.
d) Amidases - cleave amide bonds. Examples: arginase, urease, glutaminase, etc. Many amidase enzymes are found in
4. Lyases - enzymes that are similar in function to hydrolases, however, when cleaving bonds in molecules, water is not consumed. Enzymes of this class always contain a non-protein part, for example, in the form of vitamins B1 or B6.
a) Decarboxylases. These enzymes act on the C-C bond. Examples are glutamate decarboxylase or pyruvate decarboxylase.
b) Hydratases and dehydratases - enzymes that catalyze the reaction of splitting C-O bonds.
c) Amidine-lyases - destroy C-N bonds. Example: arginine succinate lyase.
d) P-O lyase. Such enzymes, as a rule, cleave off the phosphate group from the substrate substance. Example: adenylate cyclase.
The biochemistry of enzymes is based on their structure
The abilities of each enzyme are determined by its individual, unique structure. Any enzyme is, first of all, a protein, and its structure and degree of folding play a decisive role in determining its function.
Each biocatalyst is characterized by the presence of an active center, which, in turn, is divided into several independent functional areas:
1) The catalytic center is a special region of the protein, along which the enzyme is attached to the substrate. Depending on the conformation of the protein molecule, the catalytic center can take a variety of forms, which must fit the substrate in the same way as a lock to a key. Such a complex structure explains what is in the tertiary or quaternary state.
2) Adsorption center - acts as a "holder". Here, first of all, there is a connection between the enzyme molecule and the substrate molecule. However, the bonds formed by the adsorption center are very weak, which means that the catalytic reaction at this stage is reversible.
3) Allosteric centers can be located both in the active center and over the entire surface of the enzyme as a whole. Their function is to regulate the functioning of the enzyme. Regulation occurs with the help of inhibitor molecules and activator molecules.
Activator proteins, binding to the enzyme molecule, accelerate its work. Inhibitors, on the contrary, inhibit catalytic activity, and this can occur in two ways: either the molecule binds to the allosteric site in the region of the active site of the enzyme (competitive inhibition), or it attaches to another region of the protein (noncompetitive inhibition). considered more efficient. After all, this closes the place for the binding of the substrate to the enzyme, and this process is possible only in the case of almost complete coincidence of the shape of the inhibitor molecule and the active center.
An enzyme often consists not only of amino acids, but also of other organic and inorganic substances. Accordingly, the apoenzyme is isolated - the protein part, the coenzyme - the organic part, and the cofactor - the inorganic part. The coenzyme can be represented by carbohydrates, fats, nucleic acids, vitamins. In turn, the cofactor is most often auxiliary metal ions. The activity of enzymes is determined by its structure: additional substances that make up the composition change the catalytic properties. Various types of enzymes are the result of a combination of all the listed factors of complex formation.
Enzyme regulation
Enzymes as biologically active substances are not always necessary for the body. The biochemistry of enzymes is such that they can harm a living cell in case of excessive catalysis. To prevent the harmful effects of enzymes on the body, it is necessary to somehow regulate their work.
Since enzymes are of a protein nature, they are easily destroyed at high temperatures. The process of denaturation is reversible, but it can significantly affect the work of substances.
pH also plays a big role in regulation. The greatest activity of enzymes, as a rule, is observed at neutral pH values (7.0-7.2). There are also enzymes that work only in an acidic environment or only in an alkaline one. So, in cellular lysosomes, a low pH is maintained, at which the activity of hydrolytic enzymes is maximum. If they accidentally enter the cytoplasm, where the environment is already closer to neutral, their activity will decrease. Such protection against "self-eating" is based on the features of the work of hydrolases.
It is worth mentioning the importance of coenzyme and cofactor in the composition of enzymes. The presence of vitamins or metal ions significantly affects the functioning of some specific enzymes.
Enzyme nomenclature
All enzymes of the body are usually named depending on their belonging to any of the classes, as well as on the substrate with which they react. Sometimes, not one, but two substrates are used in the name.
Examples of the names of some enzymes:
- Liver enzymes: lactate dehydrogenase, glutamate dehydrogenase.
- Full systematic name of the enzyme: lactate-NAD+-oxidoreduct-ase.
There are also trivial names that do not adhere to the rules of nomenclature. Examples are digestive enzymes: trypsin, chymotrypsin, pepsin.
Enzyme Synthesis Process
The functions of enzymes are determined at the genetic level. Since a molecule is by and large a protein, its synthesis exactly repeats the processes of transcription and translation.
The synthesis of enzymes occurs according to the following scheme. First, information about the desired enzyme is read from DNA, as a result of which mRNA is formed. Messenger RNA codes for all the amino acids that make up the enzyme. Regulation of enzymes can also occur at the DNA level: if the product of the catalyzed reaction is sufficient, gene transcription stops and vice versa, if there is a need for a product, the transcription process is activated.
After the mRNA has entered the cytoplasm of the cell, the next stage begins - translation. On the ribosomes of the endoplasmic reticulum, a primary chain is synthesized, consisting of amino acids connected by peptide bonds. However, the protein molecule in the primary structure cannot yet perform its enzymatic functions.
The activity of enzymes depends on the structure of the protein. On the same ER, protein twisting occurs, as a result of which first secondary and then tertiary structures are formed. The synthesis of some enzymes stops already at this stage, however, to activate the catalytic activity, it is often necessary to add a coenzyme and a cofactor.
In certain areas of the endoplasmic reticulum, the organic components of the enzyme are attached: monosaccharides, nucleic acids, fats, vitamins. Some enzymes cannot work without the presence of a coenzyme.
The cofactor plays a decisive role in the formation Some of the functions of enzymes are available only when the protein reaches the domain organization. Therefore, the presence of a quaternary structure is very important for them, in which the connecting link between several protein globules is a metal ion.
Multiple forms of enzymes
There are situations when it is necessary to have several enzymes that catalyze the same reaction, but differ from each other in some parameters. For example, an enzyme can work at 20 degrees, but at 0 degrees it will no longer be able to perform its functions. What should a living organism do in such a situation at low ambient temperatures?
This problem is easily solved by the presence of several enzymes at once, catalyzing the same reaction, but operating under different conditions. There are two types of multiple forms of enzymes:
- Isoenzymes. Such proteins are encoded by different genes, consist of different amino acids, but catalyze the same reaction.
- True plural forms. These proteins are transcribed from the same gene, but peptides are modified on the ribosomes. As a result, several forms of the same enzyme are obtained.
As a result, the first type of multiple forms is formed at the genetic level, while the second type is formed at the post-translational level.
Importance of enzymes
In medicine, it comes down to the release of new drugs, in which the substances are already in the right quantities. Scientists have not yet found a way to stimulate the synthesis of missing enzymes in the body, but today drugs are widely available that can temporarily make up for their deficiency.
Various enzymes in the cell catalyze a wide variety of life-sustaining reactions. One of these enisms are representatives of the group of nucleases: endonucleases and exonucleases. Their job is to maintain a constant level of nucleic acids in the cell, removing damaged DNA and RNA.
Do not forget about such a phenomenon as blood clotting. Being an effective measure of protection, this process is under the control of a number of enzymes. The main one is thrombin, which converts the inactive protein fibrinogen into active fibrin. Its threads create a kind of network that clogs the site of damage to the vessel, thereby preventing excessive blood loss.
Enzymes are used in winemaking, brewing, obtaining many fermented milk products. Yeast can be used to produce alcohol from glucose, but an extract from them is sufficient for the successful flow of this process.
Interesting facts you didn't know
All enzymes of the body have a huge mass - from 5,000 to 1,000,000 Da. This is due to the presence of protein in the molecule. For comparison: the molecular weight of glucose is 180 Da, and carbon dioxide is only 44 Da.
To date, more than 2,000 enzymes have been discovered that have been found in the cells of various organisms. However, most of these substances are not yet fully understood.
Enzyme activity is used to produce effective laundry detergents. Here, enzymes perform the same role as in the body: they break down organic matter, and this property helps in the fight against stains. It is recommended to use a similar washing powder at a temperature not exceeding 50 degrees, otherwise the denaturation process may occur.
According to statistics, 20% of people around the world suffer from a lack of any of the enzymes.
The properties of enzymes have been known for a very long time, but only in 1897 people realized that not the yeast itself, but an extract from their cells, could be used to ferment sugar into alcohol.
The mechanism of d-I: the main component of the walls of bacteria - peptidoglycan - a substrate for lytic enzymes lysis of the shell of microorganisms
Lysosubtilin
Formosorb
Lysozyme
Pepsinorm
Indications:
prevention and treatment of diarrheal diseases in calves (all)
chronic septic conditions, burns, frostbite, conjunctivitis, aphthous stomatitis and other infectious diseases (Lysozyme).
proteolytic enzymes.
Mechanism of d-I: lyse proteins and their decay products in necrotic tissues (do not affect healthy tissues)
Trypsin
Chymotrypsin
· Chymopsin
Terrilitin
Ribonuclease + depolymerizes RNA - delays the development of RNA-containing viruses
Deoxyribonuclease + hydrolyzes DNA - delays the development of adenoviruses, herpes viruses
Collagenase - acts primarily on collagen fibers
Ellastolitin - has a proteolytic and mucolytic effect
Indications: purulent-necrotic processes (bronchopneumonia, pleurisy, burns, purulent wounds, iritis, iridocyclitis, conjunctivitis, etc.)
fibrinolytic enzymes.
Fibrinolysin
Streptoliasis
Urokinase
Alteplase and others.
Various enzyme preparations
| Ø Lidaza Ø Ronidaza | Contain hyaluronidase → breakdown of hyaluronic acid → tissue permeability Apply: locally with hematomas, arthrosis, arthritis, tendovaginitis, for the absorption of lek. ve-in, injected s / c |
| Ø Cytochrome C | Provides processes of tissue respiration Apply: with reduced tissue respiration (asphyxia of newborns, chronic pneumonia, heart failure, infectious hepatitis, intoxication, etc.) |
| Ø Penicillinase | Inactivates benzylpenicillin Apply: with allergic reactions to penicillins |
Coenzyme preparations
Cocarboxylase
riboflavin mononucleotide
· Flavinat
Pyridoxal phosphate
Cobamamid and others.
Enzyme inhibitors
Inhibitors of proteolytic enzymes
Aprotinin (contrical, trasylol, ingitril, gordox)
Selective inhibitors of fibrinolysis
Aminocaproic acid, Tranexamic acid
Anticholinesterase pre-you
Prozerin, galantamine, physostigmine
MAO inhibitors
Nialamide, Moclobemide, Selegiline
carbonic anhydrase inhibitors
Acetazolamide (diacarb)
angiotensin-converting enzyme inhibitors
Captopril, enalapril, lisinopril, etc.
xanthine oxidase inhibitors
Allopurinol
acetaldehyde inhibitors
Enzyme reactivators
Cholinesterase reactivators:
Trimedoxime (dipiroxime)
Alloxim in\m
Isonitrosin i/m
Pralidoxime chloride
Enzyme preparations
1. Enzymatic preparations of microbial synthesis
2. Enzyme preparations of animal origin
Lytic enzymes
Proteolytic Enzymes
fibrinolytic drugs (fibrinolysin, streptolyase, alteplase, etc.)
Various enzyme preparations
Coenzyme preparations
cocarboxylase, riboflavin mononucleotide, flavinate, pyridoxal phosphate, cobamamide, etc.
Enzyme inhibitors
proteolytic enzyme inhibitors
Selective inhibitors of fibrinolysis
Anticholinesterase pre-you
MAO inhibitors
Enzyme reactivators
Enzyme preparations
Stimulating the processes of digestion
