[Development of biotechnology of fermented milk product with Lactobacillus reuteri LR1 and the evaluation of its functional property in experiment in vitro and in vivo].

In this article the biotechnology of the dairy product based on the probiotic strain of Lactobacillus reuteri LR1 is presented. The following conditions of milk fermentation were screened: fermentation by monoculture of Lactobacillus reuteri LR1, fermentation by monoculture of Lactobacillus reuteri LR1 with addition of yeast extract as growth-promoting factor, and combined fermentation by Lactobacillus reuteri LR1, Lactobacillus helveticus NK1 and Streptococcus thermophilus (HTC). It had been demonstrated that after 8 hours of cultivation the number of Lactobacillus reuteri LR1 cells in the monoculture with introduced yeast extract was up to 5,9×108 CFU/cm3 whereas cell count for the monoculture without yeast extract introduction was 1,6×107 CFU/cm3. The optimized biotechnological parameters of the dairy product fermentation, which provided the required Lactobacillus reuteri LR1 cell count, were as follows: Lactobacillus reuteri LR1 starter dosage of 6%, HTC starter dosage of 3-4%, fermentation temperature of 37±1 °C, and fermentation duration of 6 hours. The developed product possessed an apparent antagonistic activity against test-cultures of such pathogenic and opportunistic microorganisms as E. coli ATCC 25922, Salmonella typhimurium, Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae. The survival of the test-cultures cultivated with the obtained dairy product on the first cultivation day was from 36 to 46% and on the second - from 8 to 20%, in comparison with the pure test-cultures. The investigation of the functional properties of the obtained dairy product was carried out by the single-factor experiment with the albino Wistar rat-stock (initial body weight 160±10 g, n=10 in each group). Its positive effect on the rats' microbiome composition and lipid exchange indices has been demonstrated. It had been shown that the administration of the obtained dairy product in the rats' diet (5 ml per day per os) during 30 days didn't cause any abnormalities in the health status and behavior of the laboratory animals of spf-category. The number and distribution of leucocytes and lymphocytes, and granulocytes in all rats' populations (intact, control and treatment) were within the normal range. Upon the introduction of the developed dairy product into the rats' diet, the increased levels of bifidobacteria, lactic acid bacteria and typical for normal rats' microflora enterobacteria were observed in the rats' microbiome. As for the biochemical indices that characterize rats' lipid metabolism, the rats consuming fermented dairy products (control and treatment) demonstrated statistically significant reduction of blood serum cholesterol level compared to the intact rat group; additionally the rats consuming the developed in this study dairy product (treatment) demonstrated statistically significant reduction of blood serum triglyceride level compared to the intact rats. Utilized in this study Lactobacillus reuteri LR1 is the first strain that was purified in Russia and characterized as useful for manufacturing of the probiotic food products, since currently only Lactobacillus reuteri strains of foreign origin can be seen on the market.

Biotransformation of Progesterone by the Ascomycete Aspergillus niger N402.

The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6ß,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6ß-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water by Steccherinum murashkinskyi laccase: insights into the reaction mechanism.

The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Šresolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

[Transformation of humic substances of highly oxidized brown coal by the basidiomycetes fungi Trametes hirsuta and Trametes maxima].

The ability of the white rot basidiomycetes Trametes hirsuta and Trametes maxima to transform coal humic substances (HS's) under the conditions of solid phase cultivation in the presence or absence of an easily available source of corbon (glucose) has been studied. It was shown that during the growth of the fungal strains used in media, containing HS's, destructive and condensation processes of HS transformation proceeded simultaneously. Based on a comparative physicochemical analysis of the initial HS's and HS's transformed by the fungi, it was established that, despite the introduction of glucose may favor a deeper transformation of HS's by basidiomycetes, the general direction of their modification is dominant reduction or oxidation and is determined by the physiological biochemical peculiarities of the strain used.

[Comparative analysis of the ligninolytic potential of basidiomycetes belonging to different taxonomic and ecological groups].

Screening of the ligninolytic activity of basidiomycetes from the Culture Collection of the Komarov Botanical Institute, Russian Academy of Sciences, belonging to diterent taxonomic and ecological groups was performed. The patterns of the position of taxa of active producers of ligninolytic enzymes in the modern system of fungi were identified. Cluster analysis showed that the group of fungi with the greatest lign- inolytic and degradation potential includes representatives of the families Pleurotaceae, Polyporaceae, and Phanerochaetaceae, which perform the first stages of wood decomposition. As a result, species of interest for the further study of their oxidative potential and use in biotechnology were selected.

Purification, biochemical characterization, and structure of recombinant endo-1,4-ß-xylanase XylE.

The gene xylE encoding endo-1,4-ß-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding ß-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ(1/2) of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 µmol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 Å resolution. The 3D structure of an endo-1,4-ß-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.

[A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens].

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.

[Extremophilic microorganisms: biochemical adaptation and biotechnological application (review)].

The analysis of modern data on biochemical adaptation of microorganisms for living in extreme conditions is presented in this review. Special attention has been paid to the analysis of adaptive responses of microorganisms to the conditions of increased radiation at the molecular and cellular levels. The data on the practical application of extremophils and extremoenzymes, synthesized by them, biologically active substances, biopolymers and so on, have been systematized.

Effect of solvent phase transitions on enzymatic activity and structure of laccase from Coriolus hirsutus.

The effect of solvent phase transitions on catalytic activity and structure of the active site of laccase produced by the Basidiomycetes Coriolus hirsutus 072 was studied. As shown by small-angle X-ray scattering, laccase exists in solution as a mixture of monomeric and aggregated particles in the percent ratio 85:15. This ratio did not change on phase transitions. A complex nature of laccase activity dynamics during thawing and further heating to 20 degrees C was shown. Spontaneous oxidation of T1 copper center in the temperature range 12-20 degrees C was not observed. According to spectral data, the structure of laccase active sites including all copper centers of types T1, T2, and T3 changes during the phase transition.

[Characterization of plant phenolic compounds by cyclic voltammetry].

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (Eap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The Eap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (Ep/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one- and two-electron oxidation are proposed.

[Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes].

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.

[Comparison of the efficiency of polygalacturonase and beta-glucosidase enzyme preparations in stabilization of cherry plum wine material].

Pektofoetidin and Pectinex, enzyme preparations with the highest polygalacturonase and beta-glucosidase activities, were covalently immobilized on DEAE cellulose and Aminosilochromes 10 and 30. After treatment of cherry plum wine material with the soluble and immobilized enzyme preparations, the content of phenolics increased by 26 and 40%, respectively. The increase was accompanied by a decrease in the protein content (by up to 37%), carbohydrate content (by 17% on the average), and antioxidant activity (5-37%). The most efficient treatment involved Pektofoetidin immobilized on Aminosilochrome 10. It increased the clarity of the wine material and its antioxidant activity by 100 and 10%, respectively.

Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026.

The cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042-0.046 h(-1)) was observed at an initial pH value of 8.5-9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1 h(-1) at pH 8-8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84 kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6-9, with more than 70% and less than 50% of maximal activity being retained at pH 9.2 and 5.0, respectively.

Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases.

The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.

[Milk angiogenin (Review)]. / Angiogenin moloka (Obzor).

Recent data on angiogenin, a multifunctional member of pancreatic RNase protein superfamily, are summarized. Advances in the investigation of angiogenin structure, function, and properties are analyzed. Potentialities in natural angiogenin production from inexpensive dairy by-products are demonstrated.

[Milk ultrafiltrate as a promising source of angiogenin]. / Molochnyi ul'trafil'trat--perspektivnyi istochnik angiogenina.

The use of membrane technologies in the production of soft cheese (children's food) is associated with the appearance of up to 80% of angiogenin in the ultrafiltrate. An electrophoretically homogeneous preparation of angiogenin (MW approximately 17 kDa) was obtained from milk ultrafiltrate by two-stage ion-exchange chromatography. The yield of the angiogenin was approximately 60%, which corresponds to a 586-fold purification of the raw material. The obtained preparation retained stability in the course of lyophilization and could be stored at 4 degrees C for a long time without decomposition.

[Natural ether oils in the treatment of chronic pharyngitis in children in pediatric practice]. / Opyt primeneniia natural'nykh éfirnykh masel pri khronicheskom faringite u detei.

39 children aged 9-15 years had the diagnosis chronic pharyngitis (CP). The children received combined treatment which comprised ultrasound inhalations of natural ethereal oils. The 5-10 min procedure was performed once a day for 10 days. Relief of CP subjective symptoms was observed after 8-10 procedures, general condition improved. Natural ethereal oils in ultrasound inhalations proved effective in combined treatment of CP in children. The inhalations can be performed in outpatient setting.

[Expression of the genome of Mu-like phage D3112 specific for Pseudomonas aeruginosa in Escherichia coli and Pseudomonas putida cells]. / Ekspressiia genoma Mu-podobnogo faga D3112, spetsifichnogo dlia Pseudomonas aeruginosa, v kletkakh Escherichia coli i Pseudomonas putida.

The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.

[Integration of the genome of the Mu-like Pseudomonas aeruginosa bacteriophage D3112 into plasmid RP4 and its hybrid plasmid transfer into Pseudomonas putida and Escherichia coli C600 bacteria]. / Integratsiia genoma Mu-podobnogo bakteriofaga D3112 Pseudomonas aeruginosa v plazmidu RP4 i vvedeneie v sostave gibridnoi plazmidy v bakterii Pseudomonas putida i Escherichia coli C600.

The genome of a Mu-like bacteriophage D3112 specific for Pseudomonas aeruginosa was integrated in vivo into the RP4 plasmid. The fact of integration has been proved by two experiments: 1. The loss of RP4 plasmid is accompanied by loss of D3112 prophage; 2. Transfer of the plasmid by conjugation from Pseudomonas aeruginosa into bacteria of other species - P. putida PgG1 or Escherichia coli C600 leads to the occurrence of clones of these species which liberate phage capable of growing on the lawn of P. aeruginosa bacteria. The integrated state of D3112 inserted into RP4 is stable in P. aeruginosa, E. coli, P. putida. The transfer frequency of RP4 with integrated D3112 prophage into different bacteria which do not contain homoimmune prophage is essentially lower than that of the RP4 having no D3112 prophage. Specific manifestation of D3112 genome activity in E. coli cells is the sensitivity of cell growth to lower temperature (30 degrees C) of incubation.