Applied Biochemistry and Microbiology最新文献

The metabiotic properties of six Lactobacillus acidophilus strains, included in symbiotic starter cultures for the production of probiotic lactic acid products, have been studied. The antimicrobial activity spectrum of these strains in relation to contaminants that can develop during fermentation was studied. The time of cultivation of strains required for the maximum accumulation of cells and antimicrobial metabolites was determined. Activity was compared against test microorganisms of living lactobacillus cultures and lysates formed during fermentation or under stressful conditions of the gastrointestinal tract. The bactericidal activity of a number of strains in the lysates was up to 40% higher than in the culture broth. Fungicidal activity was manifested at the later stages of the lactobacilli growth. The most active strains can be recommended as probiotic cultures with metabolic properties suitable for inclusion in complex starter cultures when creating functional food and pharmaceutical products.

As a result of cloning of the inuA, inu1, aglC, and fopA genes encoding endoinulinase (endoINU), exoinulinase (exoINU), α-galactosidase C (AGLС) and sucrase (SUC), respectively, into the recipient strain Penicillium verruculosum B1-537 (ΔniaD), recombinant producer strains were obtained that are capable of producing target recombinant enzymes with a high yield (32‒50% of the total extracellular protein). Enzyme preparations of endoINU, exoINU, AGLC, and SUC were obtained and characterized. Using chromatographic methods, endoINU, exoINU, SUC, and AGLC with a molecular weights of 62, 56, 67, and 76 kDa, respectively, were isolated in a homogeneous form (according to polyacrylamide gel electrophoresis). The homogeneous endoINU had a high specific activity against Jerusalem artichoke inulin (56 U/mg). ExoINU was active towards inulin (17 U/mg), sucrose (850 U/mg), raffinose (41 U/mg), and stachyose (15 U/mg). SUC decomposed sucrose (10.5 U/mg), raffinose, and stachyose (3.8 and 1.4 U/mg, respectively). AGLC had raffinase and stachyase activities (31 U/mg and 30 U/mg, respectively), exhibited no activity towards sucrose, but had a high level of activity towards the synthetic substrate, p-nitrophenyl-α-D-galactoside (311 U/mg). The kinetic parameters (k_cat and K_m) of the hydrolysis of the corresponding substrates by homogeneous enzymes were determined. The temperature optimum was 50‒55°C for endoINU, 55‒65°C for exoINU, 65°C for AGLC, and 35°C for SUC. EndoINU, exoINU, AGLC and SUC exhibited its maximum activity at pH 6.5, 4.5, 4.5‒5.0, and 5.5‒6.0, respectively. The thermal stability of the enzymes was studied at different temperatures. EndoINU exhaustively hydrolyzed inulin with the formation of fructooligosaccharides with a degree of polymerization of 3‒8. ExoINU quantitatively converted inulin into glucose-fructose syrup (GFS) with a Glu : Fru ratio of 1 : 3, and sucrose into GFS with a Glu : Fru ratio of about 1 : 0.63 (SUC provided the same results in the sucrose hydrolysis). Soy galactooligosaccharides (raffinose and stachyose) were converted to sucrose and monosaccharides (glucose, galactose, and fructose) under the action of AGLC. The combined action of SUC, and AGLC resulted in a complete conversion of raffinose, stachyose and sucrose to monosaccharides. The same results were achieved using ExoINU. This enzyme can be considered promising for biotechnological applications due to its broad substrate specificity, which allows it be used both for the production of GFS from inulin and sucrose, and for the destruction of soybean galactooligosaccharides.

The effect of riboflavin-induced collagen photocrosslinking on the scaffold printing accuracy, namely on the area of formed niches and filament thickness, as well as on the degradation time and biocompatibility of scaffolds, has been studied. The thickness of the filaments in the riboflavin-modified scaffolds was 13–29% less and the niche area was 23–40% larger than in the control group without riboflavin. The riboflavin addition reduced weight loss and increased the scaffold degradation time in the buffer solution and collagenase solution by 1.2–1.3, and 1.4–2.0 times, respectively, depending on the time of exposure to UV light. The existence of an optimal illumination interval was established. Cultivation of mesenchymal stem cells on a riboflavin-crosslinked scaffold for a week did not lead to a decrease in their viability and proliferation rate.

The features of osteogenic differentiation of mesenchymal stem cells (MSCs) of dogs, native and genetically modified, have been compared when cultivated in different media. It was shown that calcium deposition into the extracellular matrix occurred within 21 day of cultivation of MSCs on a commercial induction medium. The use of α-MEM-based medium, which contains osteoinducing components, also caused osteogenic differentiation of MSCs, but calcium deposition was less active. Gene modification of canine MSCs with a plasmid carrying the gene for canine (Canis familiaris) bone morphogenetic protein 2 gene (BMP2) stimulated the release of alkaline phosphatase by cells, but not calcium deposition. Consequently, the formation of osteocytes from MSCs depends not only on endogenous, but also exogenous factors, which include dexamethasone in combination with ascorbic acid and β-glycerophosphate, and full osteogenic differentiation of MSCs in vitro requires many osteoinductive factors.

This review is devoted to topical problems in the purification of iron-bearing groundwater associated with the influence of biological factors. Uncontrolled processes of activation of microbial complexes and their resistance to extreme (stressful) environmental conditions lead a decrease in the specific groundwater flow rate, intensification of biocolmatation, and deterioration of the organoleptic properties of water. As a result, these processes negatively affect the functioning of the entire water-treatment system. It has been established that the formation of biofilms in the terrestrial system of the treatment of iron-containing groundwater is due to the emergence of protective mechanisms in underground microbial consortia in response to stressful technogenic factors (aeration, UV irradiation, and disinfection).

Abstract

The identification of risks associated with novel agricultural products of plant origin obtained via genome editing is an important aspect of genetic engineering. An extensive discussion is currently ongoing worldwide to clarify the similarities and differences between the “old” risks of “classic” GM plants and the “new” ones associated with genome editing, the lack of existing methods for identification and assessment of new risks. We propose here the concept of “safe by design” as applied to protection that is a new interesting tool that introduces good known standards of safety into plant bioengineering. This approach states that design options are identified to minimize or prevent risks and off-target of genome editing at the concept stage. The correlation between experimentally determined and in silico predicted off-target gRNA activity is a major challenge in the CRISPR system application. Today the most studies are focused on efficiency of gRNA design, while we pay attention specifically to the bioinformatics search and study of potential promoters, as the potential risk associates with a possible unplanned change in the transcriptional activity of promoters. We conveyed these strategies in the form of a risk assessment framework for regulation of new genetic technologies.

Strains of Bacillus genus were isolated from soil samples in the permafrost region (Yakutia, Russia). The phenotypic characteristics of the strains are given. The analysis of the obtained data made it possible to assign them to the Bacillus cereus complex. PCR analysis made it possible to determine the profile of B. cereus toxin synthesis genes in the genomes of the studied strains. Genetic characterization was obtained by RAPD genotyping and using MLVA loci used for genotyping of the anthrax pathogen. The results of genotyping at different levels of resolution made it possible to differentiate the studied strains from the B. anthracis species and to show their intraspecific genetic differences and the degree of relationship. Whole genome sequencing was carried out, based on the data of which MLST genotyping was carried out, which revealed two known sequence types and one new one that is described for the first time in this work. The results we obtained are of practical importance and are extremely interesting from the point of view of the evolution and phylogeography of the B. cereus group, since the fact that strains were isolated from permafrost suggests that their age may be much older than expected.

Abstract

The recent advent of third-generation sequencing technologies brings promise for better bacterial identification. However, the commercial applications of the current sequencing technologies are still constrained since their high cost and error rate of this sequencing technology. Thus, strategies for time-saving bacterial identification with low cost and high accuracy are urgently needed. In this study, we evaluated the performance of the Nanopore long-read sequencer and the Illumina platform alone or in combination with different data volume (0.5–80×) after assembly, to ascertain whether the incomplete genomes enable accurate bacterial identification. The results showed that the average nucleotide identity value of hybrid assembly and Illumina assembly does not change much as the amount of data increases. In addition, the ANI value reached a plateau at 5×, while the ANI values of Nanopore-only assemblies increased in the range of 5–30×. Since the assembly process was time-consuming, this study compared the accuracy of the Nanopore long-read sequencer in species identification with various sets of the reads (0.5–80×), to further confirm the minimum amount of data required for accurate bacteria identification. Without assembly, it enabled rapid and accurate bacterial identification with low coverage of Nanopore raw reads (0.5×). The results of study suggested the potential of Nanopore sequencing in rapid bacteria identification using low-depth raw reads (0.5×) without assembly, and provided data basis in developing new strategy for bacterial identification.

The regulation of cellular metabolism is a topic of interest for both fundamental and applied science, as the findings can be used in various biotechnological industries. One of the universal regulatory mechanisms that affects most cellular processes is the acetylation of lysine residues in central metabolic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase. In this work, we investigated the effect of acetylation and deacetylation on the activity of both wild type and mutant E. coli glyceraldehyde-3-phosphate dehydrogenase. We found that in vitro acetylation of wild-type GAPDH by PatZ acetyltransferase increased its enzymatic activity by two-fold, while subsequent deacetylation restored the activity to initial level. For mutant forms of glyceraldehyde-3-phosphate dehydrogenase, we demonstrated that the introduction of additional acetylation sites due to mutations altered the impact of acetylation/deacetylation processes on glyceraldehyde-3-phosphate dehydrogenase activity. Our data suggest a re-evaluation of the role of acetylation in regulating glyceraldehyde-3-phosphate dehydrogenase activity and its involvement in E. coli metabolism.

Abstract

We investigated the effectiveness of antibiotics (amikacin, bedaquiline, linezolid, moxifloxacin, and rifampicin) on metabolically inert M. abscessus obtained under conditions of potassium deficiency in vitro. It was found that bedaquiline led to a significant decrease in the ability of bacteria to form colonies on dense media, but did not lead to their death, since it was shown that during cultivation in a liquid medium, they reverted to a state of active division and growth. Moxifloxacin had a bactericidal effect against metabolically inert bacteria, irreversibly and significantly reducing the number of viable cells in culture, which emphasizes the effectiveness of its use for the treatment of infections caused by M. abscessus.