Department of Biochemistry of microorganisms

After the First World War to the 30's 20 century work on the biochemistry of microorganisms in Ukraine haven’t practically made. Its began after the establishment in 1928 of the Institute of Microbiology, Academy of Sciences of Ukraine when, along with several other departments in 1932 was founded Department of Biochemistry, the head of which was Professor. S. Fomin. By 1951, the heads of department were B.I. Kagan (died in 1942), B.M. Koldaev (1945 – 1946), PE Vizier (1947 – 1948), E.T. Soren (1949 – 1950). During this period, in the department worked 5 employees. In 1951, for the management department of biochemistry of microorganisms was invited Elena Y. Rashba – doctor of medical sciences, professor, who restored the Department of Biochemistry of microorganisms and led them for years 1951-1973. Elena Y. laid the foundations of scientific trends of the Department, which include such problems as glycopolymers and enzymes both for practical use and the definition of their role in the metabolic processes. From 1975 to 1996 the department directed by honored scientist, Doctor of Biological Sciences, professor Irina Y. Zakharova. Since 1997 the department was headed by Doctor of Biological Sciences, professor Liudmyla D. Varbanets. The studies in the department are carried out by two directions, the first is the study of the lipopolysaccharides (LPS) both of phytopathogenic bacteria and new types of Enterobacteriaceae, the second line of research is the enzymes of proteolytic and glycolytic activity.

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For the first time in the world literature was esttablished the structure of O-specific polysaccharides (OPS) of LPS of phytopathogenic bacteria – Ralstonia solanacearum and Pseudomonas syringae, as well as new Enterobacteriaceae such as Rahnella aquatilis, Pragia fontium, Budvicia aquatica. The OPS have a regular structure and composed of repeating mono-, tri - and tetrahexasaccharide linear or branched chains. O-specific polysaccharides of some strains were heterogeneous and contained several types of oligosaccharides, which differ in structure and are represented by hetero-or homopolisaccharides. All structures are unique and have not been described among the structures of O-specific polysaccharides of other gramnegative bacteria. The structure of the OPS of the one B. aquatica strain was glycerine teichoic acid, in which the second carbon atom attached by β-D-glucopyranose. A similar structure has also been established by us for the polysaccharides of gram-positive phytopathogenic bacteria Clavibacter michiganensis. The peculiarities of OPS structures were used as molecular basis of research on the intraspecies serological classification schemes. Based on the O-antigenicity of LPS it was shown the immunochemical heterogeneity of the studied species: R. solanacearum, P. syringae, P. fontium, R. aquatilis and B. aquatica. First, based on the O-antigenicity of LPS, strains of species tested were distributed to serogroups. The first time the structure of P. syringae pv. phaseolicola GSPB 711core oligosaccharide has been established. This structure in some extent similar to the structure of the other two members of the genus Pseudomonas - P. aeuruginosa and P.fluorescens. Their common feature is the presence of two residues of keto-deoxyoctonic acid (KDO) and two phosphorylated heptose residues, one of which is O-acylated in position 7 by carbamoyl group, a chemotaxonomic marker of bacteria genus Pseudomonas. A unique feature of P. syringae core is the existence of two glycoforms, in one of which, as a terminal monosaccharide of outer region, serves the L-rhamnose residue, and in the second – the KDO residue.

One of the research direction is the chemical modification of LPS in order to establish the chemical determinants of their biological activity. It is shown that the acyl, phosphate, and free hydroxyl groups of lipid A are responsible for the toxicity and pyrogenicityof LPS investigated. In experiments on laboratory animals it was show that some modified (in particular succinilated) forms of LPS are able to compete with the toxic LPS for binding sites on cell membranes and block their toxic effects by reducing the production of cytokines – mediators of septic shock.

Thus, the results of structural and functional studies of LPS are the basis for: 1) the creation of intraspecies classification schemes of gram-negative bacteria, 2) the development of new approaches in systematics and taxonomy of bacteria, 3) the creation on the basis of non-toxic LPS non-pyrogenic LPS potential therapeutic means for treating endotoxic shock that was caused by gram-negative bacteria, and which are able to block the toxic effects of LPS.

Some microbial glycopolymers are able to transform other carbohydrate-containig substrates, showing the activity of the enzyme. Due to the screening of 2040 strains of microorganisms of different taxonomic groups from Ukrainian Collection of Microorganisms we obtained producers of α-L-rhamnosidase (Cryptococcus albidus, Eupenicillium erubescens, Penicillium commune), α-amylase (Bacillus subtilis, Aspergillus flavus var. oryzae), α-galactosidase, α-N-acetylgalactosaminidase (Penicillium canescens, P. commune, A. niger). Enzymes purified to homogeneity, showed their glycoprotein nature, Physico-chemical properties, substrate specificity, functional groups of the active site, which are directly involved in catalysis have been studied. It give a possibility to reveal that the α-galactosidase, which is capable of hydrolyzing -1,4 and -1.6-links can be used for hydrolysis of difficulty digested carbohydrates of soy milk, improving the quality of soy products and also to increase the yield of sugar from beet molasses. Established specificity of α-galactosidase and α-N-α-acetylgalactosaminidase to -1, 3-galactose and -N-acetylgalactosamine, respectively, creates the possibility of using these enzymes for the transformation of red blood cells of II and III blood groups into universal donor erythrocytes. Investigations of α-L-rhamnosidase of C. albidus and E. erubescens exhibit narrow substrate specific to the p-nitrophenyl derivatives, and are able to hydrolyse only p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucoside. Specificity of the enzymes found also to natural substrates: naringin, rutin and neohesperidin. The ability of C. albidus and E. erubescens α-L-rhamnosidase to hydrolyze bioflavonoids can be used: a) for the hydrolysis of rhamnosyl residues present in the flavonoid glycosides, which improves the quality of the products that contain them b) to release from terpene rutinosides, aromatic compounds that enhance flavour of grape juices, wines and products derived from these drinks, c) for the prevention and treatment of hemorrhagic diathesis, as well as at radiation sickness d) in cosmetic and pharmaceutical industries.

A. flavus var. oryzae and B. subtilis α-amylases are able to split all kinds of starch. This makes the potential for their use in different types of industries in which the raw material is processed starch: baking, alcohol, paper industry, in the production of glucose syrups, brewing, as well as additives to detergents.

The investigations for separation, purification, estimation of properties of microbial peptidases, in particular ones which hydrolyse difficulty soluble protein substrates of animal origin (keratin, collagen, elastin, fibrin) are carried out. As a result of screening it were found the producers of such enzymes among the representatives of the genera Streptomyces, Bacillus, Yarrowia. From Streptomyces sp. 1349 and Streptomyces sp.1382 culture liquids highly active preparations of collagenase and keratinase were isolated and purified to homogeneity. The enzymes are differed in molecular weight, component composition, optimal action, sensitivity to cations and anions. Collagenase exhibits broad specificity of action on protein substrates as fibrous and globular nature (collagen, elastin, keratin, fibrin, gelatin and casein). Keratinase, conversely, hydrolysed only native keratin hair or feathers, and one of the enzymes also exhibits fibrinolytic activity. Immunochemical studies have shown that collagenase Streptomyces sp. 1349 no serological relationship with the enzyme isolated from Clostridium histolyticum ("Merk"). For the first time it was shown that inhibitors of viral proteolysis inhibit collagenase Streptomyces sp. 1349 and keratinase Streptomyces sp. 1382. These results can be used to create high-performance products for the treatment of skin diseases, which are caused by microbial collagenase and keratinase as well as in other branches of the economy.

It was shown that the bacteria Bacillus thuringiensis 27, B. sirculans 693, and a representative of the yeast Yarrowia lipolytica 2061 in submerged culture produce in the environment proteolytic enzyme complex active against casein, fibrin, haemoglobin, elastin, gelatin. The possibility to control the synthesis of enzymes by changing the duration of cultivation and composition of the culture medium was shown. By chemical mutagenesis with N-methyl-N-nitrosoguanidine was obtained strain B. thuringiensis IMV-7324 producing elastase. It was shown that the strain display also high fibrinolytic activity. A scheme for the isolation and purification B. thuringiensis peptidases with elastolytic and fibrinolytic activities was developed. The possibility of modifying peptidases to obtain practical importance of highly stable preparations by its mild immobilization on biopolymers such as cellulose and dextran, which increases the thermal stability of the enzyme have shown. An effective method of stabilization is also hydrophobic modification of enzyme molecules by using aminoacids.

The studies on structural and functional investigations of glycopolymers are widely known abroad. The results of structural studies published in the highest-rated special English-language journals. The collaborators of the department reports presentations in Canada, Portugal, Ireland, Germany, Netherlands, Poland, Hungary, and Russia. For many years joint research with institutions in Russia, in particular the Institute of Organic Chemistry of Russian Academy of Scinces are made.

The results of scientific works of the department collaborators are summarized in 8 books (3 of which were published in the last 5 years), more than 500 papers, 19 patents. Scientific investigations of department workers noted by Prize of D.K. Zabolotny (Zakharova I.J., Kosenko L.V., Varbanets L.D., 1988), and the State Prize of Ukraine in field of Science and Technology (Varbanets L.D., Zdorovenko G.M., Zakharova I.J. and others., 2009). Over the past few years has changed the composition of members of the department, not so much by age as to allow young workers were forced to research vacation. Today, the average age of the department collaborations is about 36 years. This indicates that the department has a great scientific potential for a successful future research.