Structure, Function and Biological Activity of Lipopolysaccharide Lipid A.

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all gram-negative bacteria. It consists of poly- or oligosaccharide region that is anchored in the outer membrane by a specific lipid moiety termed lipid A. Recent studies have shown that it is only the lipid A of LPS that has the function of endotoxin. Despite its general structural conservation, lipid A also has considerable structural microheterogeneity which can vary depending on diverse factors including bacterial adaptation to changing environment and external stimuli, incomplete biosynthesis, and breakdown products and/or chemical modifications. Therefore it is more appropriate to consider lipid A as a family of structurally related molecular species with different acylation and phosphorylation patterns rather than as an individual, homogeneous molecule. The studies of structure-function relationship of lipid A, which has the typical structure of E. coli type lipid A backbone, demonstrated that activities differed depending on: 1) the number of phosphoryl and acyl residues, 2) the substituted site of phosphoryl and acyl residues, 3) the chain length of acyl residues, 4) lipid A conformation. Current investigations showed that lipid A and also the integral outer membrane proteins responsible for the final stage of LPS transport are the pinpoints in solving the problem of bacterial drug resistance. The identification of inhibitors that specifically target LPS transport in vitro and more importantly in vivo have a significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.

[PURIFICATION AND PHYSICO-CHEMICAL PROPERTIES OF PENICILLIUM TARDUM a-L- RHAMNOSIDASE].

By ammonium sulfate fractionation and chromatography on TSK-gels Toyopearl HW- 60 and Fractogel TSK DEAE-650-s from supernantant of cultural liquid of Penicillium tardum 1MB F-100074 was isolated and purified in 23 times preparation of enzyme with α-L-rhamnosidase activity with yield of 3.12 %. The specific activity was 27.7 U/mg of protein, molecular mass 67 kDa, thermo- and pH optimum - 60 °C and pH 5.0 respec- tively. It was shown high pH- and thermostability of purified P tardum a-L-rhamnosidase which may be dued to presence of 12 % of carbohydrate component.

[The Effect of Metal Ions and Chemical Reagents on the Activity of Achromobacter sp. 7a α-Amylase].

The effect of cations and anions on the activity of Achromobacter sp. 7a α-amylase was studied. It is shown that tested enzyme is stable to most of anions, however sensitive to a number of cations. The most significant inhibitory effects on the activity of Achromobacter sp. 7a α-amylase exerted Hg2+, Al3+, Fe3+, Cu2+ and Ag+ ions. Decline of activity Achromobacter sp. 7a α-amylase in the presence of EDTA and EGTA indicated on the presence within its structure of metal ions. An important role in the functioning of this enzyme play a carboxyl and sulfhydryl groups as evidenced by its inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide and p-chloromercuribenzoate respectively. α-Amylase Achromobacter sp. 7a does not contain histidine imidazole group in the active center, unlike most studied glycosidases. The tested enzyme showed high stability in the presence of Tween-20, urea, peroxide of hydrogen, making it competitive with previously described α-amylases.

[Physico-Chemical Properties of Achromobacter SP. α-Amylase].

From Achromobacter sp. 7a, that was isolated with Black sea, aquatoria of island Zmi- inyi, was isolated enzyme with α-amylase activity, that able also to split the synthetic substrates: p-nitrophenyl-α-D-glucopyranoside and p-nitrophenyl-α, -ß-D-xylopyranoside. Methods of isolation and purification of enzyme were selected which included: ammonium sulfate precipitation and affinity sorption on starch, that improved enzyme activity in 7 times in comparison with activity in the supernatant of cultural liquid. a-Amylase showed maximal activity at pH 7.0 and 11.0 and to the temperature 50 °C. Enzyme remained fully stable during 24 hours in the range of pH from 7.0 to 12.0, during 3 hours at a temperature 37 °C and 50 °C at pHopt 7.0, and also 87.5 % and 75 % of initial activity saved during 3 h of incubation at a temperature 37 °C and 50 °C at pHopt 11.0 respectively. It is shown that addition of antihunt agents (ions of calcium, chloride of natrium) did not protect an enzyme from thermoinactivation (60 °C, 70 °C).

[Screening of the Producents of α-L-Rhamnosidases Amongst Representatives of Penicillium].

The aim of this work was to study α-L-rhamnosidase [КФ 3.2.1.40] - enzyme, which hydrolyse the terminal non-reduced α-1,2-, α-1,4- and α-1,6-linked L-rhamnose. As a result of screening conducted among 30 strains of micromycetes ability to synthesize α-L-rhamnosidase revealed in Penicillium tardum 60, 39, 2929, 2962, 2963, 2964, 2965, 2966, P. rugulosum 2778, 1652, 2766, P. restrictum 425, 2756, P. aculeatum 202, 217, 329, 2973, 2974, 2975, 2976, 2977, 2979 activity, which ranged from 0.07 to 0.53 OD/mg protein. The most active is brought out P. amleatum 202. From culture supernatant of this micromycete by fractionation with ammonium sulfate (90 % saturation) complex enzyme preparation was obtained and its physico-chemical properties were studied. It was shown that enzyme has pH optimum 3.0, thermooptimum - 60 °C and displayed stability in pH values from 2.0 to 4.0 during 90 min. At pH 5.0 the activity of complex enzyme preparation insignificantly decreased and appears to be up 40 % from initial one. At optimal pH value 3.0 and temperature 15 °C α-L-rhamnosidase tested was stable during 3 days. In addition to α-L-rhamnosidase enzyme preparation of P. arnleatum 202 exerted also ß-D-glucosidase activity.

[Functional and Biological Activity of Pantoea agglomerans Lipopolysaccharides].

The lipopolysaccharides (LPS) of the seven strains of Pantoea agglomerans were isolated and chemically identified. It was established that the investigated strains characterized by different relative output of LPS from 5.2 to 14.0 % by dry weight of bacteria. LPS were characterized quite high content of carbohydrates - from 22 to 54 % 2-keto-3-deoxyoctonic acid (KDO) - from 0.39 to 2.22 % and heptose - from 3.3 to 14.00 %. Fatty acids, containing in the chain of 12 to 16 carbon atoms were identified. Lipids A of all tested LPS were characterized by predominant 3-OH-C14:0 acid from 31.7 to 39.3 % depending on the strain. Since all the studied strains of P. agglomerans were sensitive to polymyxin B, it can be concluded that the LPS do not contain in the structure of lipid A, such a substitute as 4-amino-4-deoxy-L-arabinose. One of the ways of changes in the functional and biological properties of LPS is the chemical modification. As modifiers were used complexes of germanium and tin. In the study of serological activity and toxicity of modified LPS it was found that some of them lost both serological and toxic activity. It was revealed that all investigated P. agglomerans LPS decreased the median adhesion and the index of the adhesiveness. The higher concentration of P. agglomerans LPS in the reaction mixture, the less interactions between surface structures of red blood cells and E. coli cells.

[Screening of Mannane-Degrading Enzymes Producers].

The aim of research was to investigate the prevalence of complex mannan-degrading enzymes among museum and freshly isolated cultures of micromycetes, actinobacteria and bacteria. It has been shown that the producers of ß-mannanases mostly extracted from sources that are rich in plant residues. We detected strains of Penicillium aculeatum, P. tardum and P. rugulosum which produce a complex of ß-mannanases and α-galactosidase activity. Also, strains of thermophilic micromycetes species Corynascus sepedonium, Scytalidium thermophilum and Rhizomucor tauricus with high mannanase activity in culture liquid were detected (10 ­ 130 U/ml). Two strains of P. aculeatum and P. tardum showed ß-mannanase, ß-mannosidase and α-galactosidase activity. Actinobacteria was shown high potential, 70 % of all tested strains showed mannanase activity; their activity ranged from 2 to 55 U/ml. Among the most active bacterial cultures were representatives of soil microflora: Bacillus circulans, B. subtilis, B. mesentericus, where as among the strains isolated from sea water the active mannanases producers were not found.

[Screening of α-L-Rhamnosidases and Peptidases among Actinobacterium and Bacilli].

Purpose: To carry out screening of peptidases and α-L-rhamnosidases producers among actinobacterium and bacilli. Methods: The biochemical methods of α-L-rhamnosidase, elastase, caseinolytic, fibrinolytic and collagenase activity determination have been used. Results: Among 31 strains of actinobacterium and 24 strains of bacilli it was not exhibited any enzyme with elactolytic activity, while a number of actinobacterium strains displayed high collagenase activity. As to bacilli, elastolytic activity was observed only in five strains, however its level is not an interest for future investigations. Bacillus subtilis 121 and 108 exerted enough high activity (0.100 and 0.092 U/mg of protein respectively). Conclusion: The most effective producer of collagenase and α-L-rhamnosidase is actinobacterium strain 6/5 isolated from nettle zhisosphere,while peptidase with fibrinolytic activity - B. subtilis 121 and 108. We believe these strains may be perspective for further researches.

[OPTIMIZATION OF CULTIVATION CONDITIONS OF PENICILLIUM TARDUM--THE α-L- RHAMNOSIDASE PRODUCER].

The influence of some technological cultivation parameters of Penicillium tardum to synthesize of the extracellular α.-L-rhamnosidase were studied. It was shown that rhamnose (0.8%), yeasts autolysate (0.2%), temperature of the cultivation 25 degrees C, pH 5.0 are necessary for maximal α-L-rhamnosidase production. The enzyme reaches the maximal activity level in 96 hours with sulphitic number equal 0.44. At cultivation of P. tardum in the picked up conditions the α-L-rhamnosidase synthesis has raised in 4 times.

[THE EFFECT OF METAL IONES AND SPECIFIC CHEMICAL REAGENTS ON THE ACTIVITY OF ASPERGILLUS FLAVUS VAR. ORYZAE AND BACILLUS SUBTILIS α-AMYLASES].

The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.

[SEROLOGICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF PANTOEA AGGLOMERANS LIPOPOLYSACCHARIDES].

The serological and phytotoxic properties of lipopolysaccharide (LPS) of plant pathogens--Pantoea agglomerans were studied. It is known that the thin variations in the structure of the O-specific polysaccharides determining serological specificity of gram- negative bacteria and used as a molecular basis of serological classification schemes. For P. agglomerans still does not exist a classification scheme based on serology specificity of their LPS. The results of cross serological tests demonstrate immunochemical heterogeneity of species P agglomerans. Only three strains of the 8488, 8490 and 7969 according to the agglutination of O-antigens and direct hemagglutination and inhibition direct hemagglutination can be attributed to a single serogroup. Other strains--each separate group, although some have a relationship. Compared with control plants under the influence of seed treatment of LPS in plants may be reduced, and in some cases increased root length, height and weight sprout, depending on the strain from which the selected LPS. Dive seedlings of tomatoes in the solutions of the studied preparations FSC caused the loss, and after some time, restore turgor.

[ABILITY OF MICROORGANISMS FROM DIFFERENT ECOLOGICAL NICHES TO HYDROLYZE THE INSOLUBLE PROTEINS].

Screening of protease producers with specificity to insoluble and hard soluble protein substrates of animal origin (collagen, fibrin, elastin and keratin) was carried out. It was studied the bacterial cultures (24 strains) isolated from water and periphyton of enclosures with dolphins, and also from exhalations, oral cavity and skin of dolphins. Some bacterial strains isolated from water and periphyton of enclosures hydrolyzed collagen (5-23 U/ml) and elastin (20-32 U/ml). Thus all tested cultures did not possess the property of extracellular keratinases synthesis. The streptomycetes (48 strains) were isolated from the soil of Black Sea coastal strip near Odessa and Saky, from parkland and the shores of freshwater lake in Saky and from the soil of Atlantic Ocean coastal strip near Albufena (Portugal). Several streptomycetes have been found to appeare the perspective producers of extracellular keratinase and collagenase. The strains isolated from the soil of the coastal strip area both sea and freshwater lake in Saky possessed the highest activity (up to 5 U/mg).

[Glycopolymers of microorganisms: achievements and future research (review)].

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

[Component composition of Cryptococcus albidus and Eupenicillium erubescens alpha-L-rhamnosidases].

The component composition of Cryptococcus albidus and Eupenicillium erubescers alpha-L-rhamnosidases have studied. It was shown that enzymes have a monomeric structure. Enzyme preparations of C. albidus and E. erubescens have similar qualitative but differ in quantitative amino acid composition. alpha-L-rhamnosidase of C. albidus characterised by high amount of histidine, proline, cysteine, methionine in compared with alpha-L-rhamnosidase of E. erubescens. alpha-L-Rhamnosidase of E. erubescens, in contrast to the alpha-L-rhamnnosidase of C. albidus, contained higher levels of lysine, arginine, threonine, alanine, isoleucine, leucine, tyrosine, phenylalanine. It is shown that purified preparations of alpha-L-rhamnosidase C. albidus and E. erubescens contained 5 and 1% carbohydrates respectively. Enzyme preparations differ in quantitative monosaccharide composition, which represented by rhanmose, xylose, mannose, galactose and glucose. Furthermore, alpha-L-rhannosidase C. albidus contained fuicose, whereas alpha-L-rhamnosidase E. erubescens--ribose and arabinose. A significant percentage of hydrophobic amino acids, which is 31 and 34% of the total content, and the presence of the carbohydrate component are essential in stabilization of enzymes molecule.

[Phytotoxic properties of Ralstonia solanacearum lipopolysaccharides].

The study is dedicated to research of phytotoxic properties of Ralstonia solanacearum lipopolysaccharides. This causative agent is one of the most dangerous among potato bacterial diseases. It is revealed that the inhibitory effect of LPS solution on seedlings germination is more noticeable on crops susceptible to brown rot. Maximal total phytotoxic properties have been shown by LPS from strains 35, 52b, TX1 and TS3, which were characterized by relatively low rhamnose content. Relative to the control plants LPS may diminish and some ones--increase the root length, height and weight of seedlings, subject to particular strain. But the stimulation revealed is minor.

[Influence of coordination compounds of germanium (IV) and stannum (IV) on activity of some microbial enzymes with glycolytic and proteolytic action].

Influence of coordinative compounds of germanium (IV) and stanum (IV) (complexes of germanium (IV) with nicotinamide (Nad) [GeCl2(Nad)4]Cl2 (1) and complexes of stanum (IV) with 2-hydroxybenzoilhydrazone 4-dimetylaminobenzaldehide (2-OH-HBdb) [SnCl4(2-OH-Bdb-H)] (2), 3-hydroxy-2-naphtoilhydrazone 2-hydroxynaphtaldehide (3-OH-H2Lnf) [SnCl3(3-OH-HLnf)] (3) and izonicotinoilhydrazone 2-hydroxyibenzaldehide [SnCl3 (Is·H)] (4) on activity of peptidases 1 and 2 Bacillus thuringiensis, α-L-rhamnosidase Cryptococcus albidus, Eupenicillium erubescens and α-amylase Aspergillus flavus var. oryzae. Results testify that all studied compounds differ on their influence on activity of the enzymes tested: significantly don't change elastolytic activity of peptidases 1 and 2 B. thuringiensis, completely inhibit A. flavus var. oryzae amylase, activate or oppress of α-L-rhamnosidase C. albidus and E. erubescens. Considerable differences in compounds (3, 4) on activity observed in case of the last. It's possible that peculiarity of influence (1) in compare with (2-4) is connected with existence of different central atoms of complexants: germanium (IV) (1) and stanum (IV) (2-4). A certain analogy in oppression of C. albidus α-L-rhamnosidase by compounds (1) and (4) can explain with presence of a pyridinic ring at molecules of their ligands. The less activsty displayed compound (2) with coordinative knot {SnCl4ON}. Nature of compounds (3, 4) activity was absolutely different: essential increase of activity of C. albidus α-L-rhamnosidase and full oppression of E. erubescens α-L-rhamnosidase by compound (3), while the action of compound (4) was feed back. Taking into account identical coordination knot {SnCl3O2N} the major role in this case play change of a hydrazide fragment in molecules of their ligands.

[Hydrolytic activity of microorganisms of the Dead Sea coastal ecosystems].

All strains tested are characterized by proteolytic (caseinolytic) activity, while elastase one was revealed only in two Gracilibacillus strains 6T2 and 7Tl. The activity was high enough (23.1 and 34.7 E/Ml, respectively). These values are at the level of bacterial producers which are described in literature: Bacillus mesentericus 316 M (6 E/Ml), Bacillus thuringiensis IMB B-7324 (50-55 E/Ml). The ability of two strains tested to synthesize enzyme, active against elastine, is important, so far as microbial enzyme may be perspective for using in medicine: elastases are able to dissociation of elastin fibres of connective tissues. These two strains display also fibrinolytic activity, however it was insignificant. Six of eight strains studied manifested alpha-amylase activity (0.01 - 1.173 E/Ml). It was shown that no strains, isolated from the Dead Sea costal ecosystems are able to manifest alpha-L-rhamnosidase activity.

[Kinetic characteristics of alpha-L-rhamnosidase of Eupenicillium erubescens].

It was established, as a result of investigations of the alpha-L-rhamnosidase of Eupenicillium erubescens kinetic properties, that K(m) and V(max) for the corresponding synthetic substrate were 1.0 mM and 120 micromol/min/mg of protein, respectively. alpha-L-Rhamnosidase was also competitively inhibited by the reaction product- L-rhamnose, the inhibition constant was 5.2 x 10(-2) M. One could also observe the inhibition of alpha-L-rhamnosidase reaction in the presence of 10(-3) M of glucose. It was shown that the rate of enzymatic hydrolysis of nitrophenyl substrate was directly proportional to the concentration of enzyme, and the increase of the substrate concentration leads to the increase of hydrolysis rate. The substrate concentration being increased above the optimal one (5.0 mg/ml), the reaction rate decreases due to the formation of inactive enzyme-substrate complex FS2.

[Physico-chemical properties of Bacillus thuringiensis IMV B-7324 fibrinolytic peptidase].

Investigations of physico-chemical properties of the Bacillus thuringiensis IMV B-7324 fibrinolytic peptidase showed that optimal activity of enzyme displayed at pH 10.0 and temperature 60 degrees C. Stability of peptidase retained in the range of pH from 6.0 to 11.0 and temperature from 20 to 50 degrees C over 1 h. Inhibition of fibrinolytic peptidase activity by ethylene glycol tetraacetic acid (EGTA) and tetrasodium salt of ethylenediaminetetraacetic acid (trilon B) indicates the belonging of this enzyme to the group of metallopeptidases. It was established that cations Ag+, Mg2+ and Ba2+ increased the fibrinolytic activity by 40 %, 25 % and 30 %, respectively, but Ca2+, Zn2+, Cu2+, Pb2+ and Hg2+ reduced it by 30-60%. Several of studied anions (F-, Br-, SO2-, S2O(2-)3, AsO(3-)4, NO-(3) and NO2(-) inhibit the activity of B. thuringiensis IMV B-7324 fibrinolytic peptidase by 25-100%.

[Identification of 16S RNA and PagL genes of Ralstonia solanacearum].

Features of nucleic acids sequences of genes of lipid A deacylases of R. solanacearum, which are presented in GenBank database, were analyzed. Primers to this gene were selected, and using them 12 strains of R. solanacearum were analyzed for the presence of this gene in their genome by means of PCR method. PagL gene was identified in strain 749, and dependence of its transcription on some environmental conditions was established.