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Vol 60, No 4 (2024)

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Articles

Immunoassay using full-length and phage antibodies for antibiotic detection

Guliy O.I., Dykman L.A.

Abstract

The widespread use of antibiotics, leading to antibiotic resistance and the appearance of antibiotics in the environment and food, stimulates the development of new methods for monitoring antibacterial drugs in environmental objects. One of the promising areas for the development of methods for determining antibiotics belongs to sensor technologies. The key point in the development of sensory systems is the selection of a sensitive (recognizing) element. One of the most popular methods for recognizing antibiotics is the use of antibodies. The work presents the main immunosensory systems based on recording the “antigen-antibody” interaction and shows the advantages and disadvantages of polyclonal and monoclonal antibodies. The possibility of using phage antibodies to determine of antibiotics is described separately.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):325-339
pages 325-339 views

Optimization of biosynthesis of butyric acid from glucose through the inverted fatty acid β-oxidation pathway by recombinant Escherichia coli strains

Gulevich A.Y., Skorokhodova A.Y., Debabov V.G.

Abstract

The biosynthesis of butyric acid from glucose though the inverted fatty acid β-oxidation by recombinant Escherichia coli strains was optimized. The increased yield of the target compound was achieved resulting from the plasmid expression of atoB, fadB and fadE/fabI genes in the core strain MG∆4 PL-tesB ΔyciA (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, adhE, PL-SDj10-tesB, ∆yciA). The positive effect of enforced ATP hydrolysis on microaerobic conversion of carbohydrate substrate to the final product by the recombinants was demonstrated. Activation of the futile cycle of pyruvate-phosphoenolpyruvate-pyruvate, due to the increased expression of the ppsA gene, ensured a marked increase in glucose consumption by the recombinants and led to an increase in the molar yield of butyric acid up to 39.5%. When the components of the H+-ATP synthase complex were uncoupled resulting from the deletion of atpFH genes, the molar yield of butyric acid from glucose demonstrated by the strain forming butyryl-CoA by the action of enoyl-ACP reductase FabI reached 46%.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):340-347
pages 340-347 views

The effect of various domestically produced proteolytic enzyme preparations on the organoleptic properties of pea protein isolates

Kravchenko I.V., Furalyov V.A., Pshennikova E.S., Kostyleva E.V., Sereda A.S., Kurbatova E.I., Tsurikova N.V., Fedorov A.N., Popov V.O.

Abstract

The work investigated the effect of four enzyme preparations (EP): Bacillolysin, Agroprot, Protozyme and Protozyme C (Russia), on smell, taste, as well as protein and peptide profiles of protein isolate isolated from Focor peas. It has been shown that enzyme treatment improves the odor characteristics of the isolate. Thus, it was possible to significantly reduce the severity of bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to significantly reduce the severity of such disturbing flavors as legume, astringent, bitter and herbal. The results obtained made it possible to select EP (fungal acidic aspartate proteinase) to improve the organoleptic parameters of pea protein isolates intended for the manufacture of analogues of meat and dairy products.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):348-355
pages 348-355 views

Platinum nanoparticles in aqueous solutions of chitosan-vinylpyrrolidone copolymer: synthesis and biological activity

Zuev D.N., Cherkasova E.I., Apryatina K.V., Zaitsev S.D., Smirnova L.A.

Abstract

Grafted copolymers of chitosan-vinylpyrrolidone, water-soluble at a pH of 6.8–7.5, were obtained. A technique has been developed for obtaining an aggregatively stable system of platinum nanoparticles in copolymer solutions with an average size of ~ 4 nm. The thermophysical and structural characteristics of the powdered composition of a platinum nanoparticle-copolymer are investigated. An in vitro comparison of the antitumor activity of solutions of the developed composition and cisplatin at the same platinum concentration was performed. It was found that with respect to the culture of HeLa Kyoto and A431 cancer cells, the composition is five and two times less effective than cisplatin, respectively. Along with this, the biocompatibility of the composition is 17 times higher than that of cisplatin, which allows its use at elevated concentrations and the development of an antitumor agent with platinum nanoparticles commensurate in effectiveness with cisplatin.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):356-365
pages 356-365 views

The dynamics of the levels of cytoplasmic HSP70 and chloroplast HSP70B chaperones under heat stress differs in three species of pumpkin with different resistance to stress

Murtazina N.D., Sharapova L.S., Yurina N.P.

Abstract

The first line of defense in plants under stress is the cell chaperone system. In this work, we studied the effect of heat stress on the levels of cytoplasmic chaperones HSP70 and HSP70B in chloroplasts of three species of Cucurbita (C. maxima Duchesne, C. pepo L. and C. moschata Duchesne), which differ in resistance to stress. A relationship has been established between the levels of chaperones HSP70 in the cytoplasm and HSP70B in chloroplasts and the species of pumpkin plants under heat stress conditions. Under stress, a significant increase in the level of chaperones was observed in the cells of pumpkin plants C. maxima – the level of HSP70 in the cytoplasm increased by 3.6 times, and the level of HSP70B in chloroplasts – by two times. Heat stress caused a 1.7-fold increase in the level of the cytoplasmic chaperone HSP70 in the cells of C. pepo pumpkin plants, but no significant change in the level of the HSP70B protein was noted. However, as a result of the effect of heat stress on C. moschata pumpkin plants, a decrease in the levels of HSP70 and HSP70B was revealed compared to untreated plants. The dynamics of changes in the levels of chaperones in the cytoplasm and chloroplasts under the influence of heat stress are similar. It should be noted that the constitutive level of HSP70 and HSP70B under normal conditions in C. moschata and C. repo is higher than in C. maxima. Analysis of the data obtained revealed an interesting pattern: high constitutive levels of HSP lead to insignificant induction of HSP and vice versa – low constitutive level of these proteins correlates with high induction of these proteins after heat stress. The data obtained are important for understanding the mechanisms of plant resistance to stress and can be useful for the selection and creation of highly resistant productive varieties of agriculturally important plants.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):366-374
pages 366-374 views

Phylogeny and characterization of endophytic bacteria isolated from the rhizosphere of pea seedlings (Pisum sativum L.)

Makarova L.E., Markova Y.A., Zaytseva Y.V., Bychkova A.A., Gorbenko I.V., Konstantinov Y.M., Vasiliev I.A., Morits A.S., Bizikov P.A.

Abstract

We have previously shown the ability of endophytic bacteria to move out of the pea plant seedling roots (Pisum sativum L.) into the rhizosphere. In this study, six distinct bacterial strains were isolated from the root growth medium during the cultivation of seedlings in an aqueous medium. By analyzing the nucleotide sequence of 16S rRNA genes, the taxonomic position of these strains was established, their morphological and cultural parameters were assessed, the activity of hydrolytic enzymes (pectinase, cellulase, protease) and the IAA-producing capability were examined. It has been observed that the quantity of endophytic bacteria that appears on the root surface during the growth of pea seedlings significantly surpasses the quantity present in the root tissues. It is assumed that hydrolytic enzymes such as pectinase and cellulase are involved in the release of bacteria into the external environment, causing the destruction of carbohydrate structures in plant cell walls. The metabolic parameters established in the studied strains and the significance of these endophytic bacteria for the host plant after their exit from the roots into the rhizosphere are under discussion.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):375-382
pages 375-382 views

Use of strain Pseudomonas sp. OBA 2.4.1 for pre-sowing treatment of pea seeds (Pisum sativum L.) in the presence of heavy metals and glyphosphate

Khakimova L.R., Chubukova O.V., Vershinina Z.R.

Abstract

The effect of the PGPB strain of bacteria Pseudomonas sp. was studied. OBA 2.4.1, resistant to NiCl2 (up to 3 mM), Pb(CH3COO)2 (up to 5 mM) and glyphosate (up to 8 mg/ml), on Pisum sativum L. plants at different concentrations of HMs and herbicide. It was found that the strain under study had a positive effect on the length of the roots of pea plant seedlings in the presence of HM, which indicates an increase in the plant’s resistance to stress caused by exposure to nickel and lead. However, this effect was not recorded in the experimental version with the addition of glyphosate, which confirmed its high toxicity. The results obtained indicate that the strain Pseudomonas sp. OBA 2.4.1 promoted the growth of Pisum sativum L. under stress exposure to nickel and lead, which can be used in the development of complex-action biological products intended both to protect agricultural plants from the effects of heavy metals and to reclaim contaminated soils.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):383-391
pages 383-391 views

Bacterial expression of CRM197: investigation and optimization of gene expression factors for effective production in E. coli.

Rogozhkin S.O., Gerasimov A.S.

Abstract

CRM197 (Cross Reacting Material 197) is an inactive form of C. diphtheriae exotoxin used as a carrier protein for the development and production of conjugated polysaccharide vaccines and immunotherapeutic drugs. However, the development of these research areas is not possible without an efficient and cost-effective technology to produce CRM197 of the proper quality. In this study, we developed a highly efficient method to produce recombinant CRM197 as a fusion with SUMO protein, yielding more than 3 grams per liter in form of the inclusion bodies. We examined the significant effect of expression vector type, heterologous gene expression conditions and cultivation on its solubility. Using a combination of reduced cultivation temperature and the promoter of the gene encoding the heat shock protein CspA, we achieved an increase in the solubility level of SUMO-CRM197 of more than 30%, with an overall biosynthesis level of more than 2 grams per liter. Coexpression of the target gene with the DsbC disulfide isomerase gene allowed us to obtain the target protein completely in the soluble state with a yield of more than 1.4 grams per liter. The results obtained may become the basis for the development of a promising domestic technology for the production of CRM197.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):392-402
pages 392-402 views

The role of Yersinia pestis antigens in reception of the plague diagnostic bacteriophage L-413C

Byvalov A.A., Dudina L.G., Kravchenko T.B., Ivanov S.A., Konyshev I.V., Morozova N.A., Chernyadiev A.V., Dentovskaya S.V.

Abstract

The role of surface antigens of Yersinia pestis in reception of the phage L-413C was experimentally evaluated. Based on the methods of the phage inactivation after its co-incubation with the soluble or bead-bounded antigens, an importance of LPS from the plague microbe in the phage reception and inability to bind a capsular antigen F1, Ail protein and two autotransporters YapF and YapM were confirmed. The native and recombinant PsaA, being solved, significantly inhibited the lytic activity of the phage in comparison with the bead-bound antigens. The knockout EV cells (ΔpsaA) are able to bind the phage particles as well as the wild strain. The use of three methods to evaluate the role of PsaA antigen in phage L-413C reception gave contradictory results. On the one hand the reactive domains of PsaA are able to interact with phage particles in solution. At the same time, these domains appear to determine nonspecific binding of PsaA protein to the underlying bacterial cell structures and polystyrene microsphere, preventing phage adhesion.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):403-412
pages 403-412 views

Development of a method for detection and quantitative analysis of engeneered endolysin LysAm24-SMAP in biological samples

Klimova A.A., Grigoriev I.V., Vasina D.V., Anurova M.N., Gushchin V.A., Antonova N.P.

Abstract

In recent years modified bacteriophage lysins are widely investigated for the purposes of antibacterial therapy development. Thus, effective and precise methods for the quantitative analysis of these enzymes are of high demand. The enzyme-linked immunosorbent assay (ELISA) method has been developed for the detection of recombinant modified endolysin LysAm24-SMAP in biological samples. The optimal parameters for protein detection were determined, particularly, the influence of salt and the composition of the buffer system for samples preparation was studied. The applicability of the immunodetection system of the genetically engineered endolysin LysAm24-SMAP in various biological samples with enzyme concentrations from 0.4 ng/ml was demonstrated. Also, the influence of matrix effects in animals’ organs and tissues homogenates samples, producer strain lysates and their individual components during the analysis was assessed and it was shown that 0.65 M NaCl addition in the ELISA buffer is crucial for achieving correct results and reduces non-specific interactions in the case of LysAm24-SMAP. The effectiveness of the developed system in the immunochemical control of the bacteriolytic enzyme was confirmed.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):413-423
pages 413-423 views

Influence of organic solvents on the results of immunoenzyme determination of herbicide butachlor: selection of sample preparation modes

Berlina A.N., Smirnova N.I., Komova N.S., Serebrennikova K.V., Zherdev A.V., Dzantiev B.B.

Abstract

The influence of organic solvents such as methanol and acetonitrile on the results of immunodetermination of butachlor in samples of rice and rice products was studied. The schemes of enzyme immunoassay using: (a) native antiserum containing specific antibodies to butachlor and antispecies antibodies labeled with horseradish peroxidase, and (b) biotinylated specific antibodies and streptavidin labeled with peroxidase are considered. The close values of IC10 (0.77 and 0.68 ng/ml, respectively) and working range (2.6–165 and 2.4–192 ng/ml, respectively) were established for the two schemes, when analyzing in a mixture of phosphate buffer and methanol 85 : 15%. For the second scheme, the detection of butachlor in samples of rice and food products based on it is shown at a level of 80–132% of the administered amount of the analyte. The comparison of sample preparation methods allows us to recommend the extraction of butachlor with hexane with redissolution of the dry residue in a buffer with 15% methanol, or the preparation of a methanol extract followed by a 6-fold dilution with a buffer solution.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(4):424-432
pages 424-432 views