Applied Biochemistry and Microbiology最新文献

At present, biosensor tools for analyzing intracellular energy processes are actively being developed. In this work, a set of plasmid constructs with luciferase genes from Luciola mingrelica and Photorhabdus luminescens was proposed for obtaining biosensor cells based on bacteria and eukaryotes. The possibility of targeted expression of luciferase both in the cytoplasm and in the mitochondrial matrix was realized due to the use of signal sequences. It was demonstrated that the resulting biosensors are suitable for analyzing the cell pool of macromolecular compounds and reducing equivalents, such as ATP, NADH, and flavin mononucleotide (FMN). We hope that this tool will be in demand for the analysis of the activity of intracellular chaperones, as well as toxicological and other investigations.

The enzymes that are least represented but most in demand in biotechnology are highly specific proteases that can hydrolyze difficult-to-degrade substrates. Micromycetes from the genus Aspergillus have been identified as producers of these proteases, which have diverse biochemical properties. For Aspergillus ochraceus VKM F-4104D, which has been previously studied as a producer of a protease that activates protein C and human factor X, its ability to secrete proteolytic enzymes that act on globular and fibrillar proteins has been investigated when cultured in nine different nutrient media with varying nitrogen sources. Maximum collagenolytic and fibrinolytic activity was observed when using a medium containing bovine collagen as an inducer. During preparative isoelectric focusing and subsequent zymography, two proteases with isoelectric points of 5.6 and 6.2 and molecular weights of 74 and 33 kilodaltons, respectively, were identified. As a result of genomic analysis of A. ochraceus VKM F-4104D, 121 potential secreted proteases have been predicted. Transcriptomic analysis revealed that when grown on a medium containing bovine collagen with high proteolytic activity, the microorganism expresses 117 extracellular proteases. Of these, only two were identified using traditional enzymological methods. Thus, the use of more advanced techniques allows for the detection of previously unidentified proteases with different activities among organisms for which information on physiology has been gathered or individual enzymes have been described previously, whereas traditional methods for identifying new enzymes often yield sparse or unrepresentative results.

The conditions of recombinant ɑ-amylase production in an induction-free cultivation system based on Bacillus mojavensis are described. Fermentation was carried out in pilot fermenters under deep cultivation, which ensured the production of experimental batches in the quantities necessary for comprehensive studies of the samples obtained. Gradual accumulation of the recombinant enzyme in the bioreactor reaches a maximum after 31 hours. Some biochemical parameters of the recombinant enzyme were studied. Its thermostability was established, and the pH optimum of activity manifestation was determined—the maximum efficiency of substrate hydrolysis was observed when the sample was preheated for 30 min at a temperature of 55°C and pH equal to 7.2. An inversely proportional relationship between the change in the level of amylolytic activity and the amount of calcium chloride added during cultivation was revealed. The possibility of creating strains producing recombinant ɑ-amylase on the basis of B. mojavensis strain BDV-1 was demonstrated.

Using cryostructuring techniques, a new biocompatible, wide-porous gelatin-hyaluronate material was prepared, which was successfully tested as a carrier of a protein-peptide bioregulator. The following physicochemical parameters of the synthesized carrier were determined: the total water-holding capacity (51.2 ± 5.8 g H₂O/g polymer), the degree of swelling (7.03 ± 0.65 g H₂O/g polymer) of the polymer inside the macropore walls of the spongy material and the compressive modulus of elasticity (12.3 ± 1.5 kPa). The presence of a system of interconnected macropores with a cross section from 20 to 210 μm in the material is shown. Next, the resulting carrier was loaded with a protein-peptide bioregulator isolated from bovine sclera and used in the experiments on organ cultivation of a biological model: the posterior section of the newt eye. As a result, a significant protective effect of the scleral bioregulator included in the sponge carrier on the viability of tissues and cells of a biological model was revealed and the prospects of a new biocompatible cryogenically structured material for dosed release of the bioregulator into the culture medium was shown.

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.