Search

Previously, elastase activities have been found in some microorganisms and presented by metalloproteases, which may play an important role in helping bacterium invasiveness and establishment of infection. The elastases from Bacillus species are serine proteases, however, to date there are few reports on systematic study of these enzymes producers among representatives of Bacillus, as well as studies of their properties. The aim of this work was to study some physicochemical characteristics of partially purified elastase enzyme from a number of Bacillus sp. strains. Methods. The objects of the study were strains of Bacillus sp. (L1, L2, L9) isolated from the dry grass of the coastal zone of the Kinburn split (Mykolaiv region). Cultures were grown under submerged cultivation at 28°C, with a shaking speed of the nutrient medium of 230 rpm for 2 days. Methods of determining elastase activity in the culture liquid supernatant were used. Results. It has been shown that Bacillus sp. L9, L1, and L2 are characterized by high levels of elastase activity (35.80, 28.0, and 33.80 U/mL, respectively). The maximum synthesis of elastase occurs on the second day of cultivation of the producer at 28 °C and shaking at 230 rpm. Maximum hydrolysis of elastin by Bacillus sp. L1 and L2 are carried out at 40 °C but at different pH optima. Thus, for the preparation of Bacillus sp. L1, optimal pH is 8, and for L2 - 10. For the complex enzyme preparation of Bacillus sp. L9, maximum hydrolysis of elastin occurs at pH 9 and temperature 50 °C. [ABSTRACT FROM AUTHOR]

Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties, facile culture conditions, and ease of cell manipulation. Among microbial enzymes, proteases are ubiquitous in nature and have been found in all living forms encompassing the eukaryotes like plants, animals, fungi, and protists as well as the prokaryotic domains of bacteria and archaea. Proteases are the most important for the industry and constitute approximately 60% of the total industrial enzyme market. Among the bacteria, the genus Bacillus has a very prominent place in terms of the commercial production of proteases. Earlier from the water and bottom sediments of the Black Sea, we have isolated a number of producers of proteolytic enzymes from Bacillus species. The aim of this work was to investigate the ability of representatives of a number of soil bacilli species to synthesize enzymes that hydrolyze such protein substrates as elastin, fibrin, fibrinogen, and keratin. Methods. The objects of the study were 8 cultures (KS 1 -- KS 8) isolated from the soil of the rice agrocenosis. Cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of for the nutrient medium of 230 rpm for 4 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and keratinase) activity in the culture liquid supernatant were used. Disulfide reductase activity was measured spectrophotometrically at 412 nm by evaluating the yellow sulfide formed during the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Results. The study of the spectrum of proteolytic activities of 8 freshly isolated strains showed that only KS 6 under experimental conditions did not show the ability to hydrolyze any of the studied substrates (casein, elastin, fibrin, fibrinogen, and keratin). Strains KS 1, KS 2, KS 7, and KS 8 showed higher levels of activity compared to other strains studied. The most interesting for further research are: І) strain KS 1, which showed the highest fibrinolytic activity, ІІ) strain KS 2 as the most effective producer wiThelastase and fibrinogenolytic activity, III) KS 7 and KS 8, which simultaneously showed the highest rates as keratinase (7 U/mL and 9 U/mL) and sulfate reductase (33 μmol/min and 31 μmol/min) activity, respectively. Conclusions. According to the catalytic properties, a number of representatives of Bacillus, isolated from the soil of the rice agrocenosis may be promising for further research as an enzyme producer with proteolytic activity. [ABSTRACT FROM AUTHOR]

In 2002, Wolf described a new species of Stenotrophomonas, Stenotrophomonas rhizophila, which is a non-pathogenic plant inhabitant. The defining characteristics of the new species, in contrast to S. maltophilia, were the following: growth at 4 °C, but its absence at 37 °C; the use of xylose as a carbon source; low osmolytic tolerance. S. rhizophila is isolated exclusively from the rhizosphere or from the internal tissues of plants, in particular from the vascular tissues of the root and stem. In 2014, a number of authors, studying cultivated heterotrophic bacteria and yeasts, which dominate in plant samples collected from various terrestrial biotopes near the Ukrainian Antarctic station on Galindez Island in Marine Antarctica, isolated a number of bacterial isolates. The authors' phylogenetic analysis using only BLAST analysis made it possible to determine the approximate phylogenetic affiliation of the bacterial isolate to the family Gammaproteobacteria (genus Stenotrophomonas, species S. rhizophila). It is known that many processes that determine the biological features of microorganisms, the nature of their relationships between them, as well as micro- and macroorganisms in biocenoses, are carried out with the participation of the surface structures of the bacterial cell, which are in direct contact with the environment. Among them, of particular interest are lipopolysaccharides (LPS), components of the outer membrane of Gram-negative bacteria. However, to date, we have not found any works devoted to the study of S. rhizophila lipopolysaccharides in the literature available to us. The aim of the work was to clarify the phylogenetic position of the 6p5m bacterial strain isolated from the Antarctic region by constructing dendrograms, to study some of its phenotypic properties, to isolate lipopolysaccharides and study chemical and component composition, serological, and biological properties. Methods. Identification was carried out via ribosomal phylogeny. A fragment of the 16S rRNA gene of bacterial strain 6p5m was amplified by PCR using primers 8F and 1492R. The phylogenetic position of the strains was determined by construction of dendrograms, which show the position of the investigated strains among closely related and typical species and type species (programs ClustalX 2.1, Mega v. 6.00). LPS was obtained from cells by water-phenol extraction, electrophoresis was carried out in polyacrylamide gel, monosaccharide and fatty acid composition was determined by chromato-mass spectrometry, antigenic activity was studied by immunodiffusion in agar. Results. Phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene revealed a high level of homology (99.8%) of the bacterial strain 6p5m with typical strain S. rhizophila DSM 14405T from the GenBank database, which allows us to classify it. Comparing phylogenetic analysis with phenotypic data, we can assert that strain 6p5m belongs to the S. rhizophila species. When studying the biopolymer composition of LPS by specific reactions to each component, it was found that the carbohydrate content was relatively low and amounted to 27.42% and 13.34% for LPS1 and LPS2, respectively. The content of characteristic LPS components: heptose and 2-keto-3-deoxyoctonic (KDO) acid was 1.78% and 0.034%, as well as 5.38% and 0.09%, respectively, for LPS1 and LPS2. Analysis of the monosaccharide composition of LPS preparations showed that fucose (72.57%) is present as a dominant monosaccharide in LPS1 and rhamnose (65.04%) in LPS2. The main monosaccharides of the extracellular component were galactose (39.92%) and rhamnose (24.36%). Analysis of the lipid part of the studied LPS indicates significant differences between them. In the composition of lipid A LPS1 and LPS2 of S. rhizophila 6p5m, anti-iso-pentadecanoic acid (a-i-C15:0) was predominant (25.58% and 39.24%, respectively), and 3-hydroxydodecanoic acid (3-OH-C12:0) in LPS2 (26.21%), hexadecanoic acid (С16:0) (27.06% and 10.51%, respectively) for LPS1 and LPS2. Cyclic acids were present only in LPS1, while i-C15:0 and 9-C16:1 only in LPS2. Electrophoretic analysis showed heterogeneity characteristic of an LPS molecule, manifested by a plurality of bands in the form of a «ladder» when the gel was stained with silver ions, which is associated with the presence in the composition of LPS preparations of Ospecific polysaccharide structures with different lengths of oligosaccharide chains, which determine their different molecular weight. On the electropherogram of LPS1 and LPS2, bands were not visualized in the upper part of the electrophoretic track, which indicates the absence of S-forms of LPS with long O-specific chains, but a significant predominance of SR-form molecules in the membrane LPS pool. S. rhizophila 6p5m LPS showed significantly higher pyrogenic activity compared to pyrogenal, a pharmaceutical preparation, an active ingredient of which is Shigella typhi LPS. The extracellular polymer did not exhibit pyrogenic activity. In the double immunodiffusion reaction in agar according to Ouchterlony, it was found that the studied LPSs in the homologous system exhibit antigen activity. The antigens present in both LPS1and LPS2 and the extracellular component are identical: the antigen present in each of the wells binds to all antibodies that can interact with the antigen from the other well. It was shown that pre-sowing treatment of seeds with LPS preparations of S. rhizophila 6p5m stimulates the energy of seed germination. LPS1 (+2.82 cm) showed the greatest impact in comparison with the control. A significant stimulatory effect of LPS1, LPS2, and extracellular polymer was observed when studying their effects on mustard seedlings. Conclusions. The taxonomic position of bacterial strain 6r5m of the ecosystems of the polar region (Antarctica) was determined via the phylogenetic analysis taking into account the phenotypic features of the strain. The bacterial strain is represented in the phylum Proteobacteria, class Gammaproteobacteria, family Xanthomonadaceae, genus Stenotrophomonas, species S. rhizophila. From S. rhizophila cells, LPS preparations were obtained, which differed in monosaccharide and fatty acid composition, but showed high pyrogenicity. S. rhizophila represents a promising alternative to S. maltophilia for applications in agricultural biotechnology and biological control due to its ability to both stimulate plant growth and protect roots from biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

The specifics of the processing of livestock and poultry products is that in the process of obtaining the main marketable products, about half the feedstock at various stages of the technological process turns into waste that pollutes the environment. These by-products contain large amounts of the hard-to-digest keratin protein. The use of specific enzymes capable of degrading this protein helps not only to reduce the negative anthropogenic impact on nature but also to obtain valuable hydrolysates that can be used as a fertilizer for plants or a feed additive. The aim of this work was to study the ability of Bacillus megaterium UCM B-5710 to split various keratin-containing substrates: black and white chicken feathers, white turkey feathers, parrot feathers of various colors, sheep wool, pig bristles, and baby hair and nails. Methods. The culture was grown under conditions of submerged cultivation at 40 °C, with a nutrient medium stirring rate of 201 rpm for 6 days. For growth, a basic nutrient medium containing 0.5% defatted chicken feathers or other keratin-containing substrates as sole sources of carbon and nitrogen were used. Keratinase activity was assessed by UV absorption at 280 nm of hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method, caseinolytic (total proteolytic) activity was determined by the Anson method modified by Petrova, and amino acid content was determined by the ninhydrin method. The degree of hydrolysis of the substrates was evaluated by the ratio of the initial and final weight of the substrate. Results. It was shown that the synthesis of keratinase by the culture of B. megaterium UCM B-5710 begins from the 6th hour of cultivation. The level of protein and proteolytic activity and the content of amino acids increased throughout the entire period of culture growth. The supernatant of the culture liquid of B. megaterium UCM B-5710 was most effective in splitting white chicken's and turkey's feathers, a little slower -- feathers of black chicken and blue parrots, as well as wool of white sheep. According to the degree of splitting, the substrates used can be arranged in the following order: white turkey feathers > white chicken feathers > black chicken feathers > blue parrot feathers > white sheep wool > baby nails > pig bristle > baby hair. The study of the effect of feather color on the resistance to decomposition showed that black, blue, and red feathers are more resistant, which coincides with the literature data. Conclusions. B. megaterium UCM B-5710 produces keratinase capable of splitting both α- and β-keratins, however, with different efficiencies and rates. [ABSTRACT FROM AUTHOR]

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans' producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase (α-L-rhamnoside-rhamnohydrolase EC 3.2.1.40) showing specificity for terminal α-1,2-, α-1,4- and α-1,6-linked rhamnose residues, which oft en present in glycoconjugates and synthetic glycosides, can be successfully used in biotechnology for the hydrolysis of rhamnopyranoside residues present in some bioflavonoids, glycoproteins, glycolipids, and other glycoconjugates. Previously, we have shown that a significant part of the coordination compounds of various metals act as effectors of the activity of α-L-rhamnosidases. The aim of this investigation was to study the effect of a number of newly synthesized coordination compounds of Ge(IV) and Ba(II), (Co(II), Ni(II), Cu(II), Zn(II) with gluconic acid on the activity of Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. Methods. The objects of the study were Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. α-L-Rhamnosidase activity was determined by the Davis method using naringin as a substrate. Coordination compounds Ge(IV) and Ba(II), Co(II), Ni(II), Cu(II),and Zn(II) with gluconic acid were used as enzyme activity modifiers. The synthesized complexes correspond to the formulas [M(H2O)6][Ge2(OH)2(C6H8O7)2]⋅nH2O (M = Ba(1), n = 2; Co 2, n = 4; Ni(3), n = 4; Cu(4), n = 4; Zn(5), n = 3). Results. The effect of coordination compounds 1-(5) on the activity of α-L-rhamnosidase in two strains of Penicillium tardum and Eupenicillium erubescens was studied depending on the exposure time and concentration of the effector. It was shown that compound (3) at a concentration of 0.01% (1 h incubation) led to a slight (by 5%) increase in the activity of P. tardum α-L-rhamnosidase. Compound 1 at a concentration of 0.1% led to a decrease in the activity of P. tardum α-L-rhamnosidase by 29% during the first hour, and after 24 h of incubation, a decrease in the inhibitory effect to 15% was noted. Compounds 2 and (4) activated the enzyme by 9-39% at 1h exposure. At a concentration of 0.1% and exposure time of 1 h, compound 1 increased the activity of E. erubescens α-L-rhamnosidase by 80%, while at a decrease in concentration to 0.01%, the activity increased only by 29%. In general, it should be noted that in most cases, an increase in the duration of incubation up to 24 h led to a decrease in the level of activation (or inhibition) and a return to the control values of enzyme activity. Conclusions. The variety of effects of metal coordination compounds on the activity of enzymes, depending on the nature of the cation and the origin of the enzyme, has been established. The involvement of Ba(II) had the greatest activating effect on the activity of E. erubescens α-L-rhamnosidase compared to other metals. [ABSTRACT FROM AUTHOR]

The intensive development of biotechnology in the last decade is largely determined by the growing requirement needs of both medicine and various industries for products of microbial synthesis, including glycosidases, in particular a-L- rhamnosidases. lheir wide use to solve current biological-medical and chemical-technological problems stimulates re- searchers to search for compounds capable of influencing their catalytic activity. Therefore, the purpose of this work was to isolate and purify a-L-rhamnosidase from a new producer of Penicillium restrictum and to investigate multi-ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine as effectors of its activity. Methods. Ihe object of the study was a-L-rhamnosidase of restrictum. Its purification was carried out by gel filtration and ion exchange chromatography on TSK-gels and Sepharose 6B. The activity of a-L-rhamnosidase was determined us- ing the Davis method with naringin as a substrate. As modifiers of enzyme activity, purposefully synthesized multi- ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine (INi(bipy)3[Ge(HCit)2] •3H 2 O (1); [Ni(phen)3 ] [Ge(HCit)2]•2H2O (2); [{Cu(bipy)2}2Ge(μ-Cit)2] •12H20 (3); [{Cu(phen)2]2Ge-(Cit)2]•13H2O (4); [Zn(bipy)3][Ge(HCit)2].•12H20 (5); [Zn(phen)3][Ge(HCit)2]•3H2O (6)), were used. Results. From the supernatant of culture fluid of P. restrictum, a-L-rhamnosidase was isolated and purified 23.1 times with a yield of 0.09%. the specific activity of the enzyme was 27.8 units/mL. The enzyme was homogeneous according to gel filtration on Sepharose 6B and had a molecular mass of 50 kDa. It was established that the considered coordination compounds are able to regulate the catalytic activity of a-L-rhamnosidase of P. restrictum. All of them manifest themselves either as a tivators or as inert substances, no inhibition was observed. In addition, the dependence of the degree of enzyme activation by the compounds on their concentration is traced and corresponds to the following series: at a concentration of 0.01% —1 > 6 =-- 5 > 3 >2 =.-- 4 and at a concentration of 0.1 % —1 > 4 > 2 > 5 = 6.3. The catalytic activity is also significantly affect-ed by the time of exposure to the compounds: at a concentration of 0.01% for lh, the activity of the enzyme at the control level was observed for all compounds, whereas at a concentration of 0.1% for 24 h,the activity increased sharply in the presence of compounds 1(300%), 6 (153%), and 2 (134%). The action of the others was at the control level. Conclusions. The obtained data on new complex metal compounds with an activating effect on microbial a-L-rhamnosidases. It has been established that compounds whose structural organization ensures the synergism of the action of all components are the most promising enzyme effectors in a series of coordination compounds of biologically active metals and ligands. [ABSTRACT FROM AUTHOR]

Th e search for new producers of α-L-rhamnosidases and the study of their properties are constantly carried out, which makes it possible to identify enzymes with unique properties. Th us, the α-L-rhamnosidases producers of marine species of microorganisms can radically diff er in habitat conditions from terrestrial ones. Previously, we have isolated the producers of α-L-rhamnosidases from a number of representatives of the Black Sea microbiota. However, the results of these studies did not allow us to isolate a strain promising for further study of the α-L-rhamnosidase synthesized by it. Th erefore, the purpose of this work was to further search for eff ective producers of α-L-rhamnosidases among the microbiota of the Black Sea obtained from its diff erent depths. Methods. Glycosidase activities were determined by the Romero and Davis methods. Results. Th e study of α-L-rhamnosidase activity in the dynamics of growth of 10 cultures isolated from the Black Sea showed that the only glycosidase activity, which was found on the third day of cultivation in six (07, 044, 050, 052, 054, 247) of ten cultures studied, was toward α-L-rhamnosidase. However, on the 5th day of cultivation, an increase (and in some strains, the appearance) of activity was noted in all tested cultures (from 0.01 to 0.12 U/mL). On the 7th day of cultivation, α-Lrhamnosidase activity in the supernatant of the culture liquid ranged from 0.02 to 0.2 U/mL. Th e highest activity (0.2 U/ mL) was found in strain 052. On the 10th day of cultivation, the maximum activity (0.55 U/mL) was noted in culture 052 and slightly lower (0.35 U/mL, 0.28 U/mL, and 0.23 U/mL) in cultures 044, 050, and 054 respectively. Cultures 051, 020, and 247, which showed the same activity (0.1 U/mL), as well as 056 (0.09 U/mL) were an order of magnitude less active. Th e minimum activity was noted in culture 046 (0.03 U/mL). In the supernatant of the culture liquid of strain 07 on the 10th day of cultivation, α-L-rhamnosidase was absent at all. Since the highest α-L-rhamnosidase activity was found in the supernatants of culture liquids of 5 strains (044, 052, 054, 056, and 247), partially purifi ed complex preparations of those α-L-rhamnosidases were obtained for further research. Th e study of the substrate specifi city of complex enzyme preparations of α-L-rhamnosidases of strains 044, 051, 052, 056, and 247 on natural fl avonoids, such as naringin, neohesperidin, and rutin, indicated that α-L-rhamnosidase obtained from strain 052 showed the highest activity on three investigated substrates: rutin, naringin (0.55 U/mL), and neohesperidin (0.52 U/mL). In addition to natural substrates, complex preparations of α-L-rhamnosidases of strains 052, 054, 056, and 247 also hydrolyzed synthetic derivatives of monosaccharides, such as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside. Th e maximum activity (0.15 U/ mL) was noted in strain 052, whereas strain 044 was unable to hydrolyze synthetic substrates. Conclusions. Complex enzyme preparations of α-L-rhamnosidase obtained from strain 052 are promising for further investigations. Th ey showed the highest activity both on three natural substrates, such as rutin, naringin, and neohesperidin, as well as on such synthetic derivatives of monosaccharides as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside. [ABSTRACT FROM AUTHOR]

In recent years, the particular interest of researchers is focused on such enzymes as α-L-rhamnosidase and α-galactosidase. These enzymes are considered useful for various applications. α-L Rhamnosidases may be applied for debittering of citrus fruit juices, due to the less bitter taste of the derhamnosylated flavonones, for rhamnose production, and for the determination of the anomeric configuration in polysaccharides, glycosides and glycolipids. These enzymes may enhance wine aroma and flavonoid bioavailability, or assist in the synthesis of pharmaceuticals. α-Galactosidase finds application in many areas. It is widely used in the food industry to improve the quality of soy products by hydrolyzing indigestible galactosides such as raffinose and stachyose, in the processing of raw materials in order to increase the yield of sugar from molasses, and for the biotransformation of human blood erythrocytes of group B (III) in universal donor erythrocytes, as well as in enzyme therapy of some congenital disorders of sphingolipid metabolism. Earlier, as a result of screening microorganisms of different taxonomic groups, we has selected active α-L-rhamnosidase and α-galactosidase producers. One way to increase their activity is using various effector compounds capable of modifying the enzyme activity. The study of the influence of various effectors is one of the priority areas of modern research in biochemistry, biocoordination chemistry, and biotechnology. Recent advantages in the area of biocoordination chemistry revealed high activating properties of double heterometallic mixed-ligand coordination compounds with germanium(IV)/tin(IV) tartaric complex anions and 1,10-phenanthroline/2,2'-bipyridine d-metallic cations. The aim is to estimate the enzyme-effector activity of five similar tartratostannates for the α-L-rhamnosidases of Cryptococcus albidus, Eupenicillium erubescens, and α-galactosidase of Penicillium restrictum. Methods. The activity of α-Galactosidase was determined using p-nitrophenyl-α-D-galactopyranoside («Sigma», USA) as a substrate. The activity of α-L-rhamnosidase was determined using the Davis method. As modifiers of enzyme activity, [Fe(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (1), [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O (2), [Ni(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (3), [Cu(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (4), and [Zn(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·6H2O (5) were used. Results. The study of the effect of complexes 1--5, which are supramolecular salts consisting of the same tartrate stannate anion (electrophilic agent) and a 1,10-phenanthroline d-metal cation (nucleophilic agent), on the Cryptococcus albidus, Eupenicillium erubescens α-L-rhamnosidases, and Penicillium restrictum α-galactosidase showed that the compounds tested had a different influence on the enzymes' activity. The catalytic activity of α-L-rhamnosidase is significantly influenced by all complexes. The effectiveness of compounds 1--5 for P. restrictum α-galactosidase was less pronounced in comparison with C. albidus and E. erubescens α-L-rhamnosidases. It was mostly at the control level. There was observed a certain pattern in the influence of complexes on α-L-rhamnosidases of Cryptococcus albidus and Eupenicillium erubescens. Compounds 2 and 5 turned out to be the most effective and activated enzymes by 500-900%. Conclusions. Compound 2 [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O is promising for further use as an effector of the α-L-rhamnosidase activity. [ABSTRACT FROM AUTHOR]

In recent years, cold-adapted enzymes are increasingly used in industrial processes such as the food, textile and beverage industries. Moreover, cold-active enzymes are usually thermolabile and can be inactivated with little heat. This is especially important in reactions where it is necessary to inactivate an enzyme after it has completed its function, while maintaining conditions that allow other enzymes involved in the reaction to function. Among these enzymes, glycosidases play an important role, which are used in medical technological processes, the food industry, biotechnology for the purification and processing of raw materials, as well as in many other areas of human activity. Therefore, the aim of this work was to study the ability of the psychrotolerant bacterium Pseudomonas mandelii U1 to produce glycosidases, in particular a-L-rhamnosidases, and also to investigate their physicochemical properties and substrate specificity. Methods. Glycosidase activities were determined by Romero and Davis methods, protein - by Lowry method. Results. The study of enzymatic activities in the dynamics of growth indicates that already on the third day of cultivation in the supernatant of the culture liquid of P. mandelii U1 α-L-rhamnosidase activity (0.09 U/mg protein) was noted. On the fifth day of cultivation, in addition to α-L-rhamnosidase (0.09 U/mg protein), β-D-glucosidase (0.09 U/mg protein) and α-D-glucosidase (0.09 U/mg protein) activities were identified. On the seventh and ninth days of cultivation, the spectrum of glycosidase activities was wider, except for α-L-rhamnosidase (0.2 and 0.16 U/mg protein, respectively), β-D-glucosidase (0.02 and 0.05 U/mg protein, respectively) and α-D-glucosidase (0.04 and 0.08 U/mg of protein, respectively), α-D-mannosidase (0.025 and 0.025 U/mg protein, respectively), α-D-fucosidase (0.025 and 0.05 U/mg protein, respectively), N-acetyl-β-D-glucosaminidase (0.025 and 0.025 U/mg protein, respectively) and N-acetyl-β-D-galactosaminidase (0.025 and 0.025 U/mg protein, respectively). Since among the studied glycosidase activities, α-L-rhamnosidase was the highest, subsequent studies were aimed at investigating its properties. It was shown that P. mandelii U1 α-L-rhamnosidase has the pH optimum of action at 5.0, and the temperature optimum - at 4 °C. Conclusions. The temperature optimum of P. mandelii U1 α-L-rhamnosidase preparation isolated from moss in Antarctica, Galindez Island, is 4 °C, the optimum pH is 5.0, the enzyme is able to hydrolyze as synthetic substrates p-nitrophenyl-α-L-rhamnopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-α-D-mannopyranoside, and natural substrates - naringin, neohesperidin and rutin, which suggests the possibility of its use in the future in food technologies, in particular in food processing and waste degradation at low temperatures. [ABSTRACT FROM AUTHOR]

Representatives of the Pseudomonas mandelii species are able to exist and multiply in places where the temperature is constantly low. The optimum growth temperature for P. mandelii is 25-30 °C, although this bacterium can grow at 4 °C but not at 37 °C. Therefore, P. mandelii is an excellent example of psychrotolerant bacterium which like psychrophilic bacteria is characterized by a number of structural and functional adaptations that facilitate survival at low temperatures. To understand these microorganisms' role in Antarctica the characterization of its biopolymers is vital. One of these biopolymers is lipopolysaccharide (LPS), composition and structure of which are diagnostically significant. This determines the aim of the work - to isolate lipopolysaccharides from the cells of Antarctic strain of P. mandelii, grown at different temperatures, to characterize them chemically, and to study their functional and biological activity. Methods. The object of the study was Pseudomonas sp. U1, isolated from moss on Galindez Island in Antarctica. Lipopolysaccharides were extracted from dried cells by 45 % phenol water solution at 65-68 °С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin, protein - by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. As a result of phylogenetic analysis (programs ClustalX 2.1, Tree view, Mega v. 6.00) it was shown that the Antarctic bacterial strain Pseudomonas sp. U1 associated with green moss has a 99.4 % homology with the type strain from the GenBank database NR024902 P. mandelii CIP 105273T. According to these data and proximity to the corresponding cluster of species, the studied isolate can be identified as P. mandelii. A characteristic feature of LPS isolated from P. mandelii cells, grown at different temperatures (20 °C and 4 °C) is their heterogeneity. This is evidenced by the data of the monosaccharide composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. The R-form of LPS is also present, which does not contain an O-specific polysaccharide chains. Structural heterogeneity is also inherent in LPS lipid A. This is evidenced by the data of the fatty acid composition. In LPS grown at 4 °C no unsaturated fatty acids were found, while such ones are synthesized in LPS of other bacteria grown in the cold, in response to a decrease in growth temperature. The study of the immunochemical properties of LPS was carried out using polyclonal O-antisera as antibodies, and LPS as antigens indicated that in homologous systems LPS exhibited serological activity. LPS obtained from P. mandelii U1 cells, grown at 20 °C, had a complex antigenic composition and gave two clear lines of precipitation at a concentration of 1 mg/mL. LPS obtained from P. mandelii U1 cells, grown at 4 °C, gave one line, which indicates their serological homogeneity. Conclusions. For the first time lipopolysaccharides were isolated from cells of P. mandelii U1, grown at 4 °C and 20 °С. A characteristic feature of these LPS is their heterogeneity. This is evidenced by the data of the monosaccharide and fatty acid composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. LPS, obtained from cells, grown at different temperatures, are differed by serological activity. [ABSTRACT FROM AUTHOR]

Copyright of Microbiological Journal / Mikrobiolohichnyi Zhurnal is the property of D.K. Zabolotny Institute of Microbiology & Virology of the National Academy of Sciences of Ukraine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Microbiological Journal / Mikrobiolohichnyi Zhurnal is the property of D.K. Zabolotny Institute of Microbiology & Virology of the National Academy of Sciences of Ukraine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Today there are no antiviral drugs of chemical nature that can completely cure virus-infected plants. The fact that their effect is limited to minimizing the pathogenic effect of viruses motivates many researchers to look for alternatives. In recent years it has been shown that lipopolysaccharides (LPS) of some bacteria, in particular representatives of the Pseudomonas genus were active against Tobacco mosaic virus (TMV). Therefore, we were interested in the additional study of LPS of phytopathogenic bacteria Pantoea agglomerans as a possible drug acting as antiviral agent. The aim of current study was to evaluate the antiviral activities of LPS obtained from phytopathogenic bacteria P. agglomerans against TMV in vitro. Methods. The antiviral activity of LPS preparations was investigated in vitro and assessed according to the inhibition percentage towards the number of local lesions in Datura stramonium leaves. P. agglomerans LPS was isolated from dry bacterial mass by phenol-water method. LPS mild acid degradation allowed to separate O-specific polysaccharide (OPS) and lipid A, which structures were identified by us earlier. The analysis of TMV and LPS interactions was carried out using a JEM 1400 transmission electron microscope (Jeol, Japan) at an accelerating voltage of 80 kV. Results. The most active were LPS preparations from P. agglomerans P324 and 8488. In vitro inhibitory efficacies of TMV infection by these LPS preparations was 59 and 60 % respectively. LPS preparations of P. agglomerans 7969, 7604 and 9637, on the contrary, were inactive. Comparative analysis of the antiviral activity of LPS structural components of two P. agglomerans P324 and 7604 strains showed that the greatest inhibitory effect on the infectivity of TMV was exhibited by P. agglomerans P324 lipid A, the antiviral activity of which practically did not differ from the activity of the LPS molecule (it was lower by 7 %). At the same time, the inhibitory effect of P. agglomerans 7604 core oligosaccharide (OG-core) against TMV was slightly higher compared to the effect of the whole LPS molecule. It can be assumed that the OG-core stimulated the defense mechanisms of plants and prevented the development of viral infection. Electron microscopic dates have shown that P. agglomerans P324 LPS at the concentration of 1 mg/ml influenced on freely located virions in the control causing "sticking" thus forming dense clusters, complexes or "bundles" of the virus. The individual structural components of P. agglomerans P324 LPS (lipid A and OG-core) did not have the same effect as a whole molecule. Conclusions. The study of the antiviral activity of LPS in the model system TMV - Datura stramonium L. plants showed that the most active were LPS preparations of only two strains of P. agglomerans (P324 and 8488) while the other seven strains were inactive. Individual structural components: lipid A from P. agglomerans P324 and OG-core from P. agglomerans 7604 decreased the infectivity of TMV by 7 and 15 % higher than the initial LPS molecule. According to electron microscopy data the virions sticked together forming the dense clusters in case of the direct LPS-virus contacting in vitro whereas in the control it was observed just a single free virus particles. A more detailed study of the effect of individual structural components will help to understand the regularities of the LPS structure effect on TMV infectivity. [ABSTRACT FROM AUTHOR]

About 2 million tons of feathers are produced annually around the world as a by-product of poultry farming. Due to the lack of funds and the complexity of processing, they have become one of the main environmental pollutants. The biodegradation of feathers by keratinolytic microorganisms has proven to be an effective, environmentally friendly and cost-effective method of bioconverting feather waste into a nutritious, balanced and easily digestible product that contains free amino acids, peptides and ammonium ions. Aim. To investigate the ability of marine actinobacteria to synthesize enzymes with keratinolytic activity and to study some of the physicochemical properties of the most active enzyme preparation. The object of the study was 10 strains of actinobacteria isolated from bottom sediments in the area of the Pradneprovsky trench of the Black Sea shelf. Methods. Caseinolytic (general proteolytic) activity was determined by the Anson method modified by Petrova, based on the quantitative determination of tyrosine, which is formed during the enzymatic hydrolysis of casein. Keratinase activity was determined by UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. The cultivation of actinobacteria was carried out in a liquid nutrient medium with the addition of defatted chicken feathers as the main source of carbon and nitrogen. Results. The ability to hydrolyze keratin was found in five cultures. Moreover, all the strains studied were practically unable to break down casein. The Acty 9 strain (12 U/ml) showed the highest keratinase activity. Additional introduction of NaCl to the nutrient medium did not have a positive effect on the enzymes synthesis. The study of the physicochemical properties of the enzyme preparation Acty 9 showed that the pH and thermooptimum were 9.0 and 60 °C, respectively. It retained 100 % of the initial activity in the range of pH 7.0-10.0 after 3 h and 95 % activity at pH 8.0 after 24 h of incubation. The studied enzyme preparation was thermostable, since it remained active for 3 h at 50 °C and 1 h at 60 °C. Conclusions. The extracellular keratinase synthesized by actinobacterium Acty 9 is promising for further research, since the enzyme is pH and thermostable and is not inferior in its physicochemical properties to those previously described in the literature. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS) are specific components of the cell envelope of gram-negative bacteria, located at the external surface of their outer membrane and performing a number of important physicochemical and biological functions. The widespread in nature are representatives of Enterobacteriaceae family. Among them there are saprotrophic, useful human symbionts, as well as causative agents of acute intestinal infections. The role of saprophytic intestinal microbiota is not limited only to its participation in the digestion process. The endotoxin released as a result of self-renewal of the cell pool of Escherichia coli partially enters the portal blood and performs antigenic stimulation of the macroorganism. In addition, a small amount of endotoxin can also be released by live gram-negative bacteria, which, given the large population of E. coli in the intestine, can create a sufficiently high concentration of endotoxin. Aim. The study of composition and biological activity of lipopolysaccharides of new E. coli strains, found in the human body. Methods. The objects of investigation were strains of Escherichia coli, isolated from healthy patients at the epidemiological center in Kharkiv. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65-68° С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin method, protein - by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Results. In all studied E. coli LPS (2884, 2890, 2892), glucose was dominant monosaccharide (40.5, 41.1, 67.3%, respectively). LPS also contained rhamnose (1.8, 22.9, 1.6%, respectively), ribose (3.5, 6.1, 3.6%, respectively) and galactose (4.1, 20.2, 18.3%, respectively). E. coli 2884 LPS also contained arabinose (1.0%) and mannose (44.8%), while E. coli strains 2890 and 2892 LPS contained heptose (9.7 and 7.8%, respectively). Lipid A composition was presented by fatty acids with a carbon chain length from C12 to C18. As the predominant components were 3-hydroxytetradecanoic (39.2-51.3%) as well as tetradecanoic (23.1-28.5%), dodecanoic (8.9- 10.9%), hexadecanoic (4.3-7.2%) and octadecanoic (1.8-2.4%) acids. Unsaturated fatty acids: hexadecenoic (2.0-17.9%) and octadecenoic (3.4-4.2%) have been also identified. It was found that octadecanoic and octadecenoic acids were absent in the LPS of 2884 and 2892 strains, respectively. In SDS-PAAG electrophoresis, a bimodal distribution typical for S-forms of LPS was observed. The studied LPS were toxic and pyrogenic. Double immunodiffusion in agar by Ouchterlony revealed that the tested LPS exhibited an antigenic activity in the homologous system. In heterologous system E. coli 2892 LPS had cross reactivity with LPS of E. coli 2890 and М-17. Since the structure of the O-specific polysaccharide (OPS) of E. coli M-17 was established by us earlier, the results of serological reactions make it possible to suggest an analogy of the E. coli 2892 and 2890 OPS structures with that of E. coli М-17 and their belonging to the same serogroup. Conclusions. The study of the composition and biological activity of LPS of new strains of Escherichia coli 2884, 2890 and 2892, isolated from the body of almost healthy patients, expands our knowledge about the biological characteristics of the species. [ABSTRACT FROM AUTHOR]

In recent years researchers have attracted their attention to such glycosidases as α-L-rhamnosidase (α-L-rhamnoside-rhamnohydrolase - EC 3.2.1.40). The substrates of their action are widespread in the plant world glycosides such as naringin, quercetrin, hesperidin, neohesperidin, and rutin, from which α-Lrhamnosidases cleave the terminal unreduced L-rhamnose residues. This specificity of α-L-rhamnosidases can be used in various industries: food - to improve the quality of drinks (reducing bitterness in citrus juices, enhancing the aroma of wines), as well as production of food additives; in the pharmaceutical industry - to improve the biological properties of bioflavonoids, in particular anti-inflammatory. A number of them are characterized by cardio- and radioprotective effects, have antioxidant, cytotoxic, antibacterial, antisclerotic properties, and are used in the complex treatment of coronary heart disease, including angina pectoris. The use of α-L-rhamnosidases in the chemical industry is associated with a reduction in the cost of rhamnose production as well as various plant glycosides and rutinosides. In the literature available to us, no data were found on the producers of α-L-rhamnosidases among the representatives of actinobacteria, which are known to synthesize a wide range of biologically active compounds, including antibiotics and enzymes. Purpose. To study the ability of actinobacteria isolated from water and bottom sediments of the Black Sea, to produce a-L-rhamnosidase, and also to study the properties of the most active producer. Methods. Glycosidase activity was determined by the Romero and Davis methods, protein - by the Lowry method. Results. The study of 12 glycosidase activities in 10 strains of actinobacteria isolated from bottom sediments of the Black Sea indicated that 6 investigated strains showed the ability to synthesize an enzyme with a-L-rhamnosidase and b-D-glucosidase activity. Studies have shown that the highest α-L-rhamnosidase activity (0.14 U/mg protein) was manifested by Acty 5 isolate with an optimum pH of 7.0 and a temperature optimum of 38° C. The enzyme preparation showed substrate specificity both for natural (rutin, naringin, neohesperidin) and synthetic (p-nitrophenyl derivatives of L-rhamnose and D-glucose) substrates. Conclusions., Promising Acty 5 isolate with high a-L-rhamnosidase and low b-Dglucosidase activity was found among marine actinobacteria. Bacteria with two enzymes activity expand the possibilities of their practical use. [ABSTRACT FROM AUTHOR]