Prikladnaâ biohimiâ i mikrobiologiâ

Публикуются оригинальные статьи по исследованиям в области биохимии и микробиологии, которые имеют или могут иметь практическое применение, в частности исследования механизмов ферментативных реакций; путей биосинтеза; строения и функций; получения низко- и высокомолекулярных биологически активных соединений (витаминов, коферментов, нуклеотидов, аминокислот, полисахаридов, липидов и т.п.); исследование механизмов фитоиммунитета; биологические способы защиты окружающей среды; новые подходы и новые методы исследования. Публикуются тематические обзорные статьи, рецензии новых книг и анализ материалов научных конференций.

Журнал основан в 1965 году.

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Vol 59, No 3 (2023)

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RNA Interference in Plant Protection from Fungal and Oomycet Infection
Maksimov I.V., Shein M.Y., Burkhanova G.F.

Phytopathogenic fungi pose a threat to food security, limiting the biological potential of agricultural crops and reducing the quality of products. New plant protection methods based on natural systemic and cellular phytoimmunity are being developed to date, where a unique mechanism, described by the term “RNA interference” (RNAi), occupies a special place. RNAi regulates the expression of target genes in a homologically dependent manner and, with the involvement of a protein complex designated as RISC (RNA-induced silencing complex), on the one hand, it protects plants from pathogens, but on the other hand, pathogens use it as a virulence factor. Cases of bilateral exchange of small RNAs between plants and pathogens affecting them through extracellular vesicles have been described. This review discusses the role of small RNAs, as well as DCL, AGO, and RdR proteins, in the infection of plants with pathogenic fungi and oomycetes, and the prospects for using RNAi in the development of environmentally friendly, modern plant protection products.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):219-234
pages 219-234 views
Effect of the Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid Through the Inverted Fatty Acid β-oxidation by Strains
Gulevich A.Y., Skorokhodova A.Y., Debabov V.G.

Using Escherichia coli MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, the derivatives capable of synthesizing adipic acid from glucose through the inverted fatty acid β-oxidation pathway were obtained. Biosynthesis of the target compound by recombinants was ensured upon the primary condensation of acetyl-CoA and succinyl-CoA by 3-oxoacyl-CoA thiolase PaaJ and the catalysis of the final reaction of the cycle by acyl-CoA dehydrogenases FadE and FabI. Deletion in the strains of sucCD genes encoding components of succinyl-CoA synthase did not increase the relative intracellular availability of succinyl-CoA for target biosynthetic reactions and did not lead to an increase in adipic acid accumulation by the recombinants. The secretion of succinic and malic acids by the strains with an impaired tricarboxylic acid cycle remained almost unchanged, indicating the activity in the cells of glyoxylate shunt reactions that compete with the cycle reactions for isocitrate, required for succinyl-CoA formation. When isocitrate lyase, malate synthases A and G, and bifunctional kinase/phosphatase isocitrate dehydrogenase were inactivated in strains due to deletion of the aceBAK operon genes and glcB, adipic acid synthesis by recombinants increased threefold and reached 0.33 mM.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):235-243
pages 235-243 views
Detection of “Non-culturable” Cells by Culture Methods
Demina G.R., Shleeva M.O., Bagaeva D.I., Vostroknutova G.V., Kaprelyants A.S.

The specific features of the reactivation of “non-culturable” (NC) Mycobacterium tuberculosis (MTB) cells for the following propagation in liquid medium were defined, as well as the approach for the quantification of these cells by culture methods was suggested. When NC cells obtained in vitro were inoculated into standard Sauton’s liquid medium, a prolonged (up to 20 days) lag-phase is observed, in which no cell propagation is occurred. During the lag-phase, dormant cells secrete substances of unknown nature that inhibit or completely arrest the reactivation and growth of cultures when initial dormant cells concentration is above 107–108 cells/mL. Addition of meat-peptone broth (3.25 g/L) into a standard Sauton’s medium with a 10-fold reduced concentration of glycerol significantly stimulated the reactivation and propagation of the initially dormant cells inoculated at a concentration above the threshold. This modification of the medium composition made it possible to detect up to 103 times more cells in the MTB population by the method of the Most Probable Number (MPN) of cells. Increased number of the detected dormant cells in the population (up to an average of 2.5 orders of magnitude) was also achieved by adding to the reactivation medium recombinant protein RpfB (5 ng/mL), a protein of the Rpf family – resuscitation promoting factor of dormant bacteria. Perhaps the action of a Rpf enzyme is related to the products of its enzymatic activity, since an increased MPN value in the dormant cells population was also observed when the products of mycobacteria peptidoglycan hydrolysis obtained by the coaction of RpfB and endopeptidase RipA were added. The addition of sonicated peptidoglycan fragments at a concentration up to 1 μg/mL had a similar effect. The obtained results may be used as approaches to optimize liquid media composition and culture conditions aiming to identify in clinical samples the pathogen of tuberculosis remain in “non-culturable” state.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):244-252
pages 244-252 views
Cane Pretreatment by Deep Eutetic Solvents to Increase its Reactivity During Enzymatic Hydrolysis with Cellulases
Semenova M.V., Vasil’eva I.S., Yaropolov A.I., Sinitsyn A.P.

Cane was pretreated with a number of deep eutectic solvents (DES) based on choline chloride (ChCl) as a hydrogen bond acceptor; among hydrogen bond donors, lactic and oxalic acids (LacA and OxA, respectively) were the most effective. Substrate pretreatment conditions (ratio of DES-components, temperature and exposure time) were optimized, leading to the highest yield of reducing sugars (RS) and glucose during subsequent enzymatic hydrolysis with cellulase preparation based on Penicillium verruculosum. It was been established that in the case of a mixture of ChCl with LacA (the molar ratio of components is 1 : 5) pretreatment should be carried out at 80°С for 24 h, and in the case of a mixture of ChCl with OxA (1 : 1) – at 80°С for 6 hours. The degree of conversion of the pretreated substrate after 48 hours of hydrolysis in the presence of the enzyme preparation (EP) B537 was 80 and 86% by absolutely dry substances for selected mixtures of ChCl/LacA and ChCl/OxA, respectively.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):253-259
pages 253-259 views
Relationship between the Composition of Root Exsudates and the Efficiency of Interaction of Wheat Plants with Microorganisms
Shaposhnikov A.I., Belimov A.A., Azarova T.S., Strunnikova O.K., Vishnevskaya N.A., Vorobyov .I., Yuzikhin O.S., Bespalova L.A., Tikhonovich I.A.

Plant roots secrete various organic substances into the rhizosphere, which are a source of nutrition for microorganisms and largely determine the nature of plant-microbe interactions. The composition of the main fractions of root exudates in ten modern varieties of wheat was determined: amino acids, organic acids and sugars. Reliable qualitative and quantitative differences between varieties for individual components of exudates were revealed, which determined the peculiarities of cultivar clustering on this trait. Relationships between exudation and the effectiveness of plant interaction with the growth-promoting rhizobacterium Pseudomonas fluorescens SPB2137 and the phytopathogenic fungus Fusarium culmorum 30 in laboratory systems, as well as with the resistance of varieties to diseases in the field, were found. The number of P. fluorescens SPB2137 in the root zone positively correlated with the amount of many amino acids, as well as maltose, secreted by the roots. The stimulating effect of rhizobacteria on root growth positively correlated with the amount of released glucose and melibiose. The relationship between the nature of root exudation and root colonization or the susceptibility of varieties to F. culmorum 30 was not found. The analysis of correlations between the incidence of wheat varieties in the field and the intensity of exudation of certain substances, as well as with the biocomposition index of amino acid exudation, was carried out. The role of root exudate components in the formation of effective plant-microbial systems is discussed.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):260-274
pages 260-274 views
Naringenin Modulates Adenylate Cyclase Activity in Planktonic Culture and Biofilm bv.
Goncharova A.M., Lomovatskaya L.A., Romanenko A.S.

The effect of naringenin on the growth of plankton culture, biofilm density, and activity of the transmembrane form of adenylate cyclase in the cells of Rhizobium leguminosarum bv. viciae has been studied. The studies performed showed that naringenin had practically no effect on the growth of the planktonic culture of these bacteria, while the density of biofilms decreased as the concentration of naringenin increased. It was shown that naringenin activated the transmembrane form of adenylate cyclase in planktonic culture cells and biofilms. Moreover, in the planktonic culture, this effect was more significanted. It is assumed that the activation of rhizobial transmembrane adenylate cyclase by naringenin is carried out using a receptor mechanism.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):275-280
pages 275-280 views
Method of DNA Extraction from Plant for Metagenomic Analysis as Example from Grape Rupr.
Kiselev K.V., Nityagovsky N.N., Aleynova О.А.

A new method for extracting DNA from plants is proposed, using the example of wild grapes Vitis amurensis Rupr., for further preparation of libraries for metagenomic analysis. The method is based on the isolation of DNA by an inexpensive CTAB method with an additional stage of DNA purification using silica spin columns (CTAB spin method). A comparative analysis of the results of metagenomic analysis of endophytes on DNA isolated using the proposed CTAB-spin method and using the commercial set ZymoBIOMICS DNA Miniprep (Zymo Research). It was found that when using the CTAB-spin method, the number of sequences of the 16S rRNA site and the diversity of bacterial genera were 2.8 and 1.2 times greater, respectively, than when using the ZymoBIOMICS kit. At the same time, the number of sequences of the internal transcribed spacer 1 (ITS1) and the biodiversity of endophytic fungi did not differ significantly during DNA extraction by two methods. Thus, the proposed method of DNA isolation for metagenomic analysis is an available and effective alternative to commercial kits for the isolation of plant DNA for new generation sequencing methods.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):281-288
pages 281-288 views
Optimization of Biolistic Transformation Parameters for
Davlekamova A.A., Zubritsky A.V., Timofeeva T.A., Yakovleva I.V., Kamionskaya A.M.

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):289-294
pages 289-294 views
Prospects for the Application of Sensor Elements for Detecting the Presence of Bacteria in Real Time
Lachinov A.N., Lachinov A.A., Karamov D.D., Galiev A.F., Kuzina E.V., Korshunova T.Y.

A study was made of the response of a sensor element based on polydiphenylenephthalide to a change in the composition of the air medium during the cultivation of Enterobacter asburiae UOM 3 bacteria in a liquid nutrient medium. Registration of changes in the resistance of the sensor element in response to volatile organic compounds released by bacterial cells was carried out by measuring the current-voltage characteristics at specified time intervals. The results showed the relationship between the number of bacteria and the change in the resistance of the sensor element, due to the release of waste products of microorganisms. With an increase in the titer of bacteria by 3 orders of magnitude, the resistance of the sensor under the influence of volatile organic compounds accumulated in the medium decreased by 2 orders of magnitude. It is assumed that sensors based on polydiphenylenephthalide can be used to determine the presence of bacteria in various materials and media.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):295-300
pages 295-300 views
Protein Antioxidant Complex of Water Extract of Larvae of Black Beetles
Ushakova N.A., Tikhonova O.V., Ambaryan A.V., Bastrakov A.I., Dontsov A.E.

An aqueous extract from the larvae of black beetles Ulomoides dermestoides grown under controlled conditions was obtained and its proteomic analysis based on one-dimensional electrophoretic separation of proteins, their trypsinolysis in a gel and subsequent chromatography-mass spectrometry was performed. Protein identification was performed using MaxQuant v. software. The Uniprot database was used to identify proteins by homology. Catalase activity was determined spectrophotometrically by hydrogen peroxide concentration decrease. Antioxidant activity was evaluated by luminol chemiluminescence quenching. The extract contained proteins that can determine the biological activity of the extract: universal regulators of cellular processes calmodulin (62%), cytochrome c-2 (13.5%), nucleoside diphosphate kinase (11.1%), the enzymatic antioxidants superoxide dismutase, catalase, glutathione S-transferase, peroxiredoxin, glutathione synthetase as well as thioredoxin, 70 and 60 kDa heat shock proteins, chitinase complex (13.4% in total). Catalase activity was 6.3 ± 1.1 µmol H2O2/min/mg protein; antioxidant activity of 1 mg protein per ml extract was equivalent to 1.36 ± 0.3 mM trolox. The prospects for practical use of the extract as a natural antioxidant complex were noted.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):301-308
pages 301-308 views
Development of Kinetic Microplate Immunoenzyme Determination of Dibutyl Phthalate
Berlina A.N., Serebrennikova K.V., Komova N.S., Zherdev A.V., Dzantiev B.B.

Phthalic acid esters are integral components of modern plastic products and packaging materials, which causes significant contamination of food products and the environment, leading to the need for simple productive monitoring methods. The article presents a rapid enzyme-linked immunosorbent assay (ELISA) for the determination of dibutyl phthalate (DBP) in fruit juices, based on the competitive interaction between free and bound antigen for the binding sites of specific antibodies. The analytical characteristics of the method were studied in various kinetic regimes of the competition stage. Optimal conditions have been established to ensure the minimum detection limit and high measurement accuracy. The duration of the competitive stage of ELISA was chosen 30 min; the range of determined concentrations of DBP was from 0.37 to 68.34 ng/mL with a detection limit of 0.08 ng/mL. The efficiency of the proposed ELISA for testing fruit juices was shown for the chosen DBP extraction mode.

Prikladnaâ biohimiâ i mikrobiologiâ. 2023;59(3):309-317
pages 309-317 views


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