Open Access Open Access  Restricted Access Access granted  Restricted Access Subscription Access

Vol 60, No 5 (2024)

Cover Page

Full Issue

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Articles

Mycoplasma: properties, detection and decontamination methods of cell cultures and viral strains (Review)

Leonovich O.A.

Abstract

Mycoplasma contamination of continuous cell cultures and collection viral strains remains a serious problem in the biotechnology industry and experimental research. The frequency of mycoplasma contamination of cultured cell lines and viruses is 15–35%, in some cases up to 80%. Mycoplasmas cause various changes in cultures contaminated by them, up to cell death, have immunomodulatory properties, and affect the yield of certain viruses propagated in cell culture. Mycoplasmas do not have a cell wall, are able to pass through a bacterial filter, have the smallest genome (≈580 kb) among bacteria, and are capable of independent reproduction and existence. These microorganisms are resistant to most antibiotics commonly used in cell culture. Derivative groups of tetracyclines and fluoroquinolones (BM-Cyclin®, Ciprobay®, Baytril®, Plasmocin®, MRA) have shown certain effectiveness in decontaminating viral strains and cell cultures from mycoplasmas. Timely, highly sensitive detection and prevention of mycoplasma infection is of great importance. For routine scanning of mycoplasma infection of continuous cell cultures and viral strains, the methods of indicator cell culture (cytochemical) and polymerase chain reaction (PCR) are recommended, for more accurate – microbiological analysis of mycoplasma colonies on a special medium.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):435-444
pages 435-444 views

Prospects of acoustic sensor systems for virus immunodetection (Review)

Guliy О.I., Zaitsev B.D., Karavaeva О.А., Borodina I.A.

Abstract

Outbreaks of viral infectious diseases in humans and animals remain one of the global problems of our time. Therefore, one of the most popular areas in applied microbiology is the development of fast and sensitive methods for viruses detection, including those based on biosensor analysis methods. The paper describes the promise of acoustic sensor systems for viruses detection. The optimal capabilities of electroacoustic sensors in viruses detection, the possibility of conducting analysis in the presence of interfering factors (viral particles and microflora) and the repeated use of sensors are shown. The presented results demonstrate the promise of using acoustic sensors to viruses detection in microbiology, medicine, and veterinary medicine.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):445-454
pages 445-454 views

Engineering of recombinant endolysin LysSi3 to increase its antibacterial properties

Antonova N.P., Grigoriev I.V., Lendel A.M., Usacheva O.V., Klimova A.A., Usachev E.V., Gushchin V.A., Vasina D.V.

Abstract

The potential of new genetically modified recombinant endolysins as antimicrobial agents against Gram-negative bacteria was investigated. A series of enzymes based on LysSi3 lysozyme-like muramidase were obtained by modifying its sequence with antimicrobial peptides of different families and recombinant expression in E. coli was demonstrated. Modification of LysSi3 resulted in increased bacteriolytic activity against the model isolate of A. baumannii and higher kinetics rate compared to the native enzyme. The cytotoxic properties of new engineered lysins were investigated with the HEK293 and HaCaT cell lines and it was shown that modification of LysSi3 with antimicrobial peptides does not significantly increase the toxic properties in vitro.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):455-464
pages 455-464 views

Antioxidant effect of carnosine and carnosine dinitrosyl iron complexes at the conditions modeling peroxidation of biomolecules

Nasybullina E.I., Kosmachevskaya O.V., Shumaev K.B., Topunov A.F.

Abstract

The antioxidant activity of carnosine and of carnosine dinitrosyl iron complexes (DNICs) was studied. A system with metmyoglobin (metMb) or gemin in combination with tert-butyl hydroperoxide (t-BOOH) was used as experimental model. Using luminol-dependent chemiluminescence method, it was shown that carnosine and carnosine DNICs effectively diminished the level of prooxidants formed by the interaction of heme groups with t-BOOH. In addition, carnosine and carnosine DNICs inhibited formation of diene conjugates arising during the oxidation of arachidonic acid in metMb―t-BOOH system. In used reaction systems, antioxidant effect of carnosine DNICs was higher than that of carnosine. Antioxidant effect of carnosine also depended on the presence of bivalent iron ions added at the concentration equivalent to their content in DNICs. These results show that the insertion of carnosine as a ligand to nitrosyl iron complexes enhances its antioxidant properties.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):465-474
pages 465-474 views

Effect of different sucrose concentrations on the biosynthesis of poly-3-hydroxybutyrate and alginate by the bacterial strain Azotobacter vinelandii 12 under different aeration conditions

Dudun A.A., Makhina T.K., Bonartsev A.P., Bonartseva G.A.

Abstract

Bacteria of the genus Azotobacter sp. produce two classes of biologically important biocompatible and biodegradable polymers – polyoxyalkanoates, which are the bacterial reserve, and alginates (ALG), which perform the function of protecting nitrogenase from oxygen. Both polymers are becoming increasingly important for use in bioengineering, pharmaceuticals and medicine, so studies of their biosynthesis and properties are currently highly relevant. The present work shows the possibility of regulating alginate and poly-3-hydroxybutyrate (PHB) synthesis by A. vinelandii 12 culture depending on the increase of sucrose concentration in the medium under different aeration conditions. At high aeration and high sucrose concentration in the medium (50 g/L), the maximum yield of free (1.08 g/L) and capsular ALG (2.26 g/L) in the medium was obtained. Under low aeration conditions, the synthesis of free ALG was completely inhibited. The maximum value of РНB synthesis was observed at medium aeration and high concentration of sucrose (50 g/l) in the medium. The maximum molecular weight (MW) of ALG was 477 kDa, while the maximum MW of PHB was much higher, reaching 1479 kDa. At low sucrose concentrations in the medium (5 to 20 g/l), capsular ALG is predominantly synthesized (up to 100% of the sum of all polymers) at all aeration levels. With increasing sucrose concentration, PHB is predominantly synthesized (68%) under low aeration conditions, an equal ratio of PHB and capsular ALG synthesis is observed under medium aeration conditions, and free ALG is actively synthesized under high aeration conditions. This work demonstrates the possibility of obtaining a selective synthesis of ALG or PHB by A. vinelandii 12 by modifying its cultivation conditions. The results obtained can be used for the development of directed biosynthesis of target products (PHB and ALG) in biotechnology.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):475-786
pages 475-786 views

Impact of neurotransmitters on the fatty acid composition and photosynthetic pigments of the green microalga Scenedesmus quadricauda

Boyang C., Chivkunova O.B., Solovchenko A.E., Lobakova E.S., Oleskin A.V.

Abstract

Apart from their functions in the nervous system of animals, neurotransmitters operate as regulatory agents and signals in diverse kingdoms of life. Some neurotrnsmitters at low concentrations have recently been revealed to exert specific effects on microalgae, predominantly functioning as algal growth stimulators. This article presents new data on the effects of such neurotransmitterss as serotonin, norepinephrine, dopamine, histamine, and acetylcholine on the fatty acid and pigment composition of the green microalga Scenedesmus quadricauda (Turp.) Breb. K-1149. It was established that acetylcholine and, to a lesser extent, histamine increased the total fatty acid content of S. quadricauda cells, whereas serotonin and dopamine decreased the fatty acid content. Acetylcholine, histamine, and norepinephrine elevated the percentage of polyunsaturated fatty acids; in contrast, serotonin and dopamine augmented the share of saturated fatty acids. Acetylcholine and, to a lesser extent, norepinephrine increased the total chlorophyll content per 1 g of dry weight in S. quadricauda while histamine decreased the chlorophyll content. Histamine also increased the chlorophyll a/chlorophyll b and carotenoid/chlorophyll ratios, which were decreased by dopamine. The data obtained apparently are of biotechnological and ecological interest. The stimulation of fatty acid accumulation and the increase in polyunsaturated species percentage was caused by the neurotransmitters acetylcholine and histamine at low (1-10 µM) concentrations, which potentially enables facilitating the biotechnological production of health-promoting preparations for therapeutic and cosmetic purposes. However, other tested neurotransmitters (dopamine and serotonin) increased the relative content of saturated fatty acids; therefore, they apparently can be used to stimulate biofuel production, since saturated fatty acid-rich lipids are advantageous raw materials for biodiesel production. The impact of neurotransmitters on microalgal fatty acid composition and photosystem components may be considered in terms of ongoing chemical interaction between microalgae and other aquatic ecosystem components that are known to produce neurotransmitters.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):487-498
pages 487-498 views

The effect of space flight factors on the interaction of Escherichia coli with bacteriophage T7

Sykilinda N.N., Lukianova A.A., Lavrikova V.V., Kutnik I.V., Panin N.V., Staritsyn N.A., Miroshnikov K.A.

Abstract

For the first time, the interaction between bacteria and bacteriophage was studied under space conditions. The model system of E. coli and bacteriophage T7 was used. The results of the interaction depended on the duration of exposure of the system to space flight factors. During the first 2 days of microgravity exposure the virus replication rate in Space was higher than on Earth. The bacteria then have adapted to space conditions and acquired resistance to the bacteriophage, which persisted for 2 days after return to Earth. Over the next three days, the sensitivity of the E. coli to the T7 bacteriophage returned to its original level.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):499-506
pages 499-506 views

ABA-degrading strains of bacteria of the genus Pseudomonas and their influence on wheat growth

Ryabova A.S., Kuzmina L.Y., Gilvanova E.A., Galimsyanova N.F., Martynenko E.V., Vysotskaya L.B., Kudoyarova G.R.

Abstract

Three new representatives of the genus Pseudomonas have been identified that are capable of utilizing abscisic acid and positively influencing the growth and development of plants. Their physiological and biochemical properties have been studied. Based on the analysis of the 16S rRNA gene, they were identified as P. veronii IB K11-1 (99.86% similarity), P. frederiksbergensis IB Ta10m (100%), strain TaE2 was assigned to Pseudomonas sp. It was found that bacteria, when growing on a mineral-salt medium with ABA, reduced the hormone content by 50-60% with an increase in population density by two orders of magnitude. In a laboratory experiment, it was shown that the introduction of bacterial biomass (108 CFU/g of substrate) into the rhizosphere of wheat plants 10 days after treatment led to a decrease in the abscisic acid content in the roots by 18–30% and an increase in plant weight by up to 30%. Thus, new strains of growth-stimulating ABA-degrading bacteria have been identified and characterized for the first time, which may be promising for the creation of biological products that increase plant resistance to biotic and abiotic stresses.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):507-513
pages 507-513 views

Effect of ethanol on the growth of the red microalga Galdieria sulphuraria

Bolychevtseva Y.V., Stadnichuk I.N.

Abstract

Polyextremophilic red microalgae of the genus Galdieria, which inhabit hot sulphur springs under conditions unusual for eukaryotes, are capable of heterotrophy. Among the dozens of exogenous organic substrates identified for Galdieria, ethanol is not mentioned as a possible energy source. As it turned out that ethanol did not alter the growth of the model species Galdieria sulphuraria when grown in the dark. In contrast, the growth of microalgae is activated in the light, despite the known cell stressor effect of ethanol. The effect of ethanol as an oxidative stress factor may be indicated by the increase in cellular respiration observed in the dark and also in the light even before the activation of photosynthesis. The marked acceleration of growth of G. sulphuraria culture in the light is most likely due to the stimulation of respiration by ethanol with generation of CO2 and its use by chloroplasts as an additional carbon substrate during the photosynthetic process. Compared to the classical organic substrate glucose, the light-induced growth of G. sulphuraria cultures in the presence of ethanol is less intense. It can be speculated that ethanol stress in light induces the system of two consecutive key enzymes in the primary alcohol metabolism chain (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase), which then leads to the eventual complete oxidation of ethanol, resulting in accelerated growth of G. sulphuraria.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):514-523
pages 514-523 views

Methods for pre-processing cane to obtain enzymative hydrolysates with high sugar content

Semenova M.V., Rozhkova A.M., Osipov D.O., Telitsin V.D., Rubtsova E.A., Kondrat’eva E.G., Vasil’eva I.S., Morozova O.V., Yaropolov A.I., Sinitsyn A.P.

Abstract

Methods of cane pretreatment (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on its subsequent enzymatic hydrolysis by cellulases and hemicellulases complexes have been studied. Substrates with the highest reactivity were obtained by exposing the cane to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59%, and the degree of conversion of polysaccharides (cellulose and hemicellulose) into monosaccharides taking into account the yield of the substrate at the pre-processing stage was 60 and 34%, respectively. After pre-treatment of the cane with acid DES, water steam, water or water/organic solution of sulfuric acid the depth of enzymatic hydrolysis was 45, 25, 20 and 11%, and the degree of polysaccharide conversion was 26, 18, 13 and 10%, respectively. The industrial enzyme preparation Agrocell Plus with a predominant content of cellobiohydrolases and endoglucanases was most effective in hydrolyzing the dust fraction of cane, as well as cane pretreated with a solution of sulfuric acid or DES (acidic or alkaline). The industrial enzyme preparation Agroxil Plus, containing endoxylanase and cellobiohydrolases, was more effective in hydrolyzing cane after hydrothermal pretreatment or alkali solution. The results of the hydrolysis of cane pretreated with acidic or weakly alkaline DES under the action of individual (homogeneous) cellulases indicate that in both cases the key enzyme was cellobiohydrolase 1.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):524-535
pages 524-535 views

Development of a bio-selecting agent based on immobilized bacterial cells with amidase activity for bio-detection of acrylamide

Protasova E.M., Maksimova Y.G.

Abstract

Actinobacteria cells Rhodococcus erythropolis 4-1 and Rhodococcus erythropolis 11-2 and Proteobacteria Alcaligenes faecalis 2, which have amidase activity, were immobilized by entrapping barium alginate and agarose into the gel structure, as well as by obtaining biofilms on thermally expanded graphite (TEG). The operational stability of such immobilized biocatalysts after storage in frozen and dehydrated form was determined, and a prototype of a conductometric acrylamide biosensor based on such a bioselective agent was developed. The most preferred method for storing immobilized cells was freezing at temperatures from –20 to –80°C; long-term storage is also possible wet at 4–25°C. It was shown that these cells were most preferable for the biodetection of acrylamide A. faecalis 2, immobilized in an agarose gel structure. An agarose gel with bacterial cells immobilized in its structure had greater mechanical strength and stability during successive cycles of conversion of acrylamide into acrylic acid compared to barium alginate gel. The mechanical strength of barium alginate gel can be enhanced by the addition of carbon nanomaterials during cell immobilization. Growing biofilms on carbon materials used for manufacturing electrodes is also promising. Biofilms of R. erythropolis 11-2 on TEG are capable of converting acrylamide into acrylic acid in more than 20 reaction cycles while maintaining at least 50% amidase activity.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):536-544
pages 536-544 views

Inducible whole-cell biosensor for detection of formate ions

Cherenkova А.А., Yuzbashev Т.V., Melkina О.Е.

Abstract

Ten strains of the yeast Yarrowia lipolytica were constructed, the genomes of which contain hrGFP gene under the regulation of the formate dehydrogenase promoters. The resulting strains can act as whole-cell biosensors for the detection of formate ions in various mediums. By visual assessment of biomass fluorescence, we selected the three most promising yeast strains. The main biosensor characteristics (threshold sensitivity, amplitude and response time) of the selected strains were measured. As a result, in terms of characteristics, the B26 strain was recognized as the most suitable for the detection of formate ions. A carbon source for the nutrient medium that does not reduce the activation of the biosensor was selected. Furthermore, we showed that unlike formate and formaldehyde, methanol practically does not induce the biosensor fluorescence response.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):545-551
pages 545-551 views

In vivo method for biotinylation of recombinant variola virus proteins

Nikitin V.N., Merkuleva Y.A., Shcherbakov D.N.

Abstract

The work implements a method for specific in vivo biotinylation of recombinant proteins M1 and B7 of the variola virus during biosynthesis in CHO-K1 cells. To do this, co-expression of the biotin ligase BirA and target genes encoding the ectodomains of the M1 and B7 proteins with a C-terminal avi-tag was carried out in CHO-K1 cells in the presence of biotin in the culture medium. The optimal biotin concentration for the expression of M1 and B7 proteins was 125 μM. The production of biotinylated recombinant proteins has been complicated by low yields. To increase the production of target proteins, low molecular weight enhancers were added to the culture medium: lithium acetate, sodium valproate and caffeine. The enhancers increased the yield of the target protein by 1.3–4.9 times and did not affect the efficiency of biotinylation. The highest yield of biotinylated protein was achieved with the simultaneous addition of a concentration of 10 mM lithium acetate and 2.5 mM sodium valproate.

Prikladnaâ biohimiâ i mikrobiologiâ. 2024;60(5):552-560
pages 552-560 views