Applied Biochemistry and Microbiology最新文献

After prolonged storage and cryopreservation, animal spermatozoa show reduced activity and fertilizing ability. Natural microvesicles are known to improve these parameters. According to our data, co-incubation of rat spermatozoa with artificial microvesicles obtained from mesenchymal stem cells using cytochalasin B exerts a positive effect on these sex cells. Incubation of microvesicles with spermatozoa causes a slower decrease in the transmembrane potential of mitochondria and reduced accumulation of lipid peroxidation products compared to the control group. As a result of such incubation, the DNA of the sex cells does not become damaged and almost no fusion of microvesicle and sperm cell membranes is observed. Thus, the presence of artificial microvesicles may improve the quality of rat sperm during storage.

A P. verruculosum strain producing Fusarium solani extracellular cutinase CUT with a high level of expression (more than 40%) was obtained by cloning the cutB gene in the recipient strain. The homogeneous CUT protein with high pNPB enzymatic activity (1070 U/mg) was purified by chromatographic methods. The homogeneous cutinase had a temperature optimum at 55°C and a pH optimum at 8. CUT retained more than 80% activity for 30 min at 50°C. About 50% activity was retained for 3 min at 60°C, and the enzyme completely lost activity in 1 min at 70°C. CUT EP had no significant phytotoxic effect, but its enzymatic activity facilitated the penetration of herbicide into plant tissues, which was expressed in a greater proportion of treated leaves with signs of glyphosate injuries. Joint application of CUT EP and herbicide resulted in a synergistic effect and allowed reducing the herbicide dosage five times, while the effectiveness of the latter decreased only two times. The selection of more effective combinations of CUT EP with glyphosate to reduce its doses without losing the exterminating effect will reduce the medical and environmental risks of using this herbicide.

Withania coagulans (Stocks) Dunal is a valuable medicinal plant with prospects for use not only in medicine, but also in the food industry. The hairy roots of withania are a more promising source of biologically active substances than the roots of plants grown in natural conditions. There is evidence of a positive effect of the compounds KNO₃, KH₂PO₄, MgSO₄, and CaCl₂ on the growth of the roots of Withania somnifera (L.) Dunal, cobalt salts on the growth of the roots of Angelica archangelica L., and increased agar concentration on the growth of the roots of A. thaliana. In this work, the effect of these compounds on the growth of hairy roots of W. coagulans was studied for the first time. It has been shown that changing the composition of the MS medium does not contribute to an increase in the dry mass of the roots. An increase in the concentration of divalent cobalt inhibits root growth, while trivalent cobalt in a concentration of up to 100 μM prolongs the viability of roots. The addition of [Co(NH₃)]₆Cl₃ can be used for long-term storage of withania root cultures without passages.

The photobiomodulation of Escherichia coli bacteria under the exposure to continuous or pulsed red laser radiation with a pulse repetition rate of 2 Hz was studied. It was found that irradiation leads to an increase in the number of individual colonies, more than twofold in case of pulsed radiation, inter alia, due to the degradation of bacterial aggregates, i.e., the initiation points of biofilm formation, without affecting the light scattering parameters of the culture. A laser-inducible signaling network conjugated with globin O₂ sensors, DgcO diguanosine monophosphate cyclase and PdeO phosphodiesterase, was constructed. It is supposed that the conformational shift induced by laser-induced local superheating of the chromophore is able to substitute oxygen in the control of globin O₂ sensors, turning off DgcO and turning on PdeO, shifting the equilibrium towards the reduction of cyclic dimeric guanosine monophosphate, bacterial aggregates and microbial biofilms, i.e., the targets for photobiomodulation in antitumor therapy.

The effectiveness of using chitosans with different characteristics (molecular weight, viscosity, and deacetylation degree) when harvesting the biomass of the cyanobacterium Arthrospira platensis by flocculation was assessed. It has been shown that the molecular weight of the polymer is the main characteristic influencing the process of biomass harvesting. The flocculation efficiency for all tested chitosan samples reached 90% within an hour of starting the process. When using chitosans with a molecular weight of 250 ± 50 kg/mol and a deacetylation degree of 65 ± 5% as a flocculant, it took less than five minutes to achieve a flocculation efficiency of 95%. From the biomass obtained as a result of flocculation, a one-stage extraction of phycobiliproteins, C-phycocyanin and allophycocyanin, was carried out. The use of chitosan during biomass harvesting did not affect the degree of extraction of phycobiliproteins, but increased the purity and antioxidant activity of the crude extract by 41–50 and 30%, respectively, relative to the control, depending on the characteristics of the chitosan. Thus, the use of the natural biopolymer chitosan in the flocculation process of a suspension of cyanobacterium A. platensis cells significantly reduces the time of biomass harvesting and increases the purity of crude phycobiliprotein extracts, which allows their use in the food and cosmetic industries, bypassing an additional stage of purification.

The synthesis of biologically active recombinant proteins and peptides in the form of active inclusion bodies (AIBs) significantly reduces production costs. Previously, we developed an original platform for the production of AIBs in Escherichia coli cells. It included the N-terminal self-aggregating peptide (SAP) L₆KD fused to the SUMO (Smt3) protein of the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. However, AIBs produced with this platform were sensitive to sonication and extrusion of cells with French press, leading to significant product loss during the cell lysate fractionation stage. To increase AIBs stability, we investigated modifications of the original SAP. we increased the hydrophobic core of SAPs and introduced additional intermolecular interactions between SAPs with disulfide or ionic bonds. The best results were obtained for peptides with an increased hydrophobic tail (L₈KD), as well as for those containing sequences of 2‒4 cysteine residues directly at the N-terminus of the hydrophobic core of SAPs (C₂L₆KD, C₃L₆KD, C₄L₆KD). Integration of cysteine residues at a distance from the hydrophobic core of SAPs (C₂G₃L₆KD, L₆KDGSC₂), as well as the formation of additional ionic bonds (KDL₆KD, KDL₈KD), did not lead to an increase in the stability of AIBs. The prposed SAP modifications did not significantly affect the synthesis of the target protein. Under sonication, the best-performing SAP variants increased AIB yield by 30‒40%. This improvement was also confirmed during cell disintegration by extrusion via French press.

The method of subcritical and supercritical ethanolysis was used to determine its impact on the degree of fermentolysis of wheat straw polysaccharides and the yield of sugars. The heat treatment of raw materials in ethanol was performed in the temperature range of 130–355°C (30 MPa, 10 min). The intensity of biomass delignification and hydrolysis of straw polysaccharides during processing is determined by the temperature. Chemical processes occurring in the wheat straw biomass under subcritical ethanolysis conditions can contribute to the enzymatic hydrolysis of polysaccharides of technical cellulose and are associated with the destruction of easily hydrolyzable pentosans and lignin. Upon the transition to the supercritical region, the intensity of hydrolysis of straw polysaccharides increases, resulting in an increase in the yield of the fraction of water-soluble compounds at 355°C to 14.5% dry mass and a decrease in the yield of insoluble straw residue (technical cellulose) to 26%. Under these conditions, cellulose is the main source of soluble carbohydrates. The low yield of technical cellulose obtained at ethanolysis temperatures above 285°C and the high content of lignin in it make it unsuitable for the enzymatic production of sugars. In the temperature range of 200–250°C, the yield of technical cellulose was 77–87% dry mass and the yield of sugars during its enzymatic hydrolysis was 27–33% dry mass. The results substantiate the possibility of using subcritical ethanolysis as an activation method for the enzymatic hydrolysis of straw cellulose.

A biotechnological method based on the use of aqueous dispersions, or colloidal solutions, of fullerene-containing compounds for the treatment of seed material, which provides growth stimulation and contributes to an increase in plant yield, has been proposed. It was found that aqueous dispersions of fullerene C₆₀ and salts of amino acid derivatives of fullerene C₆₀ possess pronounced antioxidant activity and a wide range of other properties of biological activity. The growth-stimulating effect of aqueous dispersions of fulle-rene C₆₀ and salts of amino acid derivatives of fullerene C₆₀ on plants was shown to be effective in the case of their application for pre-sowing treatment (soaking) of seeds. In field experiments on the example of spring wheat of the “Lada” variety, the increase in plant yield, weight, and number of grains in the ear, which was promoted by pre-sowing treatment of seeds in the presence of fullerene-containing aqueous dispersions, was demonstrated. The proposed biotechnological approach has prospects for effective application in agriculture for seed treatment and increasing yields of important food crops.

The isolation and identification to species of 85 strains of osmotolerant yeasts related to Ascomycota from bees and bee-associated substrates, such as honeycombs, bee bread, and honey flowers collected in the republics of the Caucasus region including Dagestan, Kabardino-Balkaria, North Ossetia, and the Chechen Republic, as well as from candied fruits and fruit leather, are reported. Due to elective selection conditions and specific substrates, the isolated yeasts did not show much species diversity. Representatives of the genera Zygosaccharomyces and Starmerella prevailed among the isolated osmotolerant yeasts. When the levels of glycerol accumulation by the representatives of the identified genera were studied, the highest glycerol production in the absence of sulfites inhibiting the fermentation process was recorded in the Y-5206 and Y-5190 strains belonging to the species Pichia kudriavzevii, namely, 5.9 and 7.8 g/L, respectively. Under the sulfite stress conditions, when the medium pH was 8.0, the highest glycerol production level of 23.5 g/L was recorded in the S. cerevisiae Y-5189 yeast. The highest rate of glucose-to-glycerol conversion (46.3%) was shown by the Zygosaccharomyces roxii yeast strain Y-5182 in the presence of sulfites. The strains of osmotolerant yeasts belonging to the species Zygosaccharomyces roxii, as well as to all species in the genus Starmerella, which are characterized by the highest glucose-to-glycerol conversion levels at a low level of ethanol production and are able to accumulate extracellular glycerol efficiently in the absence of sulfite stress, may be of potential interest for further studies aimed at developing biotechnologies for production of this alcohol.

The results of application of an original antimicrobial polymer hydrogel based on polyvinylpyrrolidone as a local depot of antibacterial drugs for the prevention of chronicization of the infectious process during hip joint prosthetics in implant-associated infection in rabbits are presented. At the first stage, intramedullary osteomyelitis of the femur of the animals was modeled by implantation of the infected limb. On the 14th day, a second surgical intervention with sanitation of the focus of infection, removal of the limb, and unipolar hip arthroplasty was performed. All rabbits were divided into two groups: experimental, with application of the experimental gel at the stage of endoprosthesis (n = 3), and control, without gel (n = 3). After withdrawal from the experiment on the 90th day, a morphological study with evaluation of changes in the soft tissues and medullary canal, as well as microbiological analysis of samples of soft tissues, bone structures, and the removed endoprosthesis stem were carried out. In the control group, Staphylococcus aureus contamination was detected in two out of three animals, while in the experimental group, such bacteria were not detected in any of the samples tested. A comparative analysis of the cellular infiltrate in soft tissues revealed that the amount of multinucleated giant cells was significantly higher in the experimental group, and the amount of neutrophylic granulocytes, as indicators of the activity of the infectious process, were significantly higher in the control group. Thus, the use of the experimental antimicrobial polymeric hydrogel allowed us to prevent the development of a chronic infectious process, despite the fact that endoprosthesis was performed during sanitation of the osteomyelitis focus and a short course of antibacterial therapy. The presence of a pronounced giant cell reaction in soft tissues requires further investigation and at a later time after surgical intervention.