Applied Biochemistry and Microbiology最新文献

Abstract—The acetylation of lysine residues in proteins is a pivotal process in numerous biological phenomena. This modification is ubiquitous across all organisms, and it is only with the advent of advanced technologies that the intricate interplay between protein acetylation and cellular metabolism in bacteria has been elucidated. This article provides a comprehensive overview of the most recent findings in the realm of protein acetylation. Collectively, these findings underscore a burgeoning interest in this area and advancements in understanding the role of protein acetylation in bacteria as the principal posttranslational modification. This underscores its significance in gene regulation, cell signaling, metabolism, and adaptive processes.

The recombinant strain based on the acidophilic yeast Schizosaccharomyces pombe with a cloned gene of heterologous lactate dehydrogenase (LDH) was used to optimize the biosynthesis of L-lactic acid. For this purpose, the effect of inactivation of pyruvate decarboxylase genes (PDC) on the synthesis of acetate, pyruvate, and ethanol (the main by-product in the synthesis of lactic acid) was studied. Using the wild S. pombe strain, we showed that the Δpdc3 and Δpdc4 deletions did not affect these indicators, while in the Δpdc1 mutant, ethanol biosynthesis was reduced and acetate biosynthesis was increased, and the Δpdc2 mutant accumulated pyruvate. The effect of deletions of the PDC1 and PDC2 genes on lactic acid biosynthesis was tested on a model S. pombe strain containing a heterologous LDH gene from Lactobacillus pentosus. It was shown that in strains with the inactivated PDC2 gene the accumulated pyruvate was poorly consumed for the biosynthesis of lactic acid even in the presence of two different recombinant lactate dehydrogenases; the introduction of the third gene of heterologous lactate dehydrogenase led to loss of viability of the strain. At the same time, in strains with the deleted PDC1 gene, the biosynthesis of lactic acid was enhanced with the introduction of additional LDH genes. The results obtained can be used in the design of industrial lactic acid producing strains.

Promoters are crucial components of yeast production strains in metabolic engineering. Due to its high activity, the promoter of the RPL25 gene, which encodes the L25 protein of the large ribosomal subunit, is a promising object for investigation and optimization. Here, to investigate the Yarrowia lipolytica pRPL25 promoter structure, a randomized library of its sequences was generated in 100 nucleotide increments. The strength of the obtained promoters was evaluated using the green fluorescent protein as a reporter. The results showed that the minimum functional length of the studied pRPL25 derivatives was 199 bp, and the optimal functional length was 497 bp. When the sequence was shortened to 400 bp, the expression level of the reporter decreased significantly. Based on the above, we propose that the 400–500 bp region of pRPL25 comprises an upstream activating sequence (UAS). The pRPL25 promoter and its derivatives potentially can be used in bioengineering to create metabolic pathways with a precise level of gene expression. Strains constructed on their basis may be more efficient producers of target compounds compared to classical expression systems such as Saccharomyces cerevisiae.

The points of enzymatic activity, quantum yields, structure, and functions of luminol chemiluminescence activated by physical activators coumarinomics ({{C}_{{314}}}) and ({{C}_{{525}}}) under the action of (CytC-CL~)complex in aqueous medium and in a nonpolar environment have been modelled. It is shown that the enzymatic activity points and quantum yields are significantly higher in the presence of the physical activator coumarin ({{C}_{{525}}}) than in the case of its own non-activated luminescence and in the case of the physical activator ({{C}_{{314}}}). The enzymatic activity depends not only on the concentration of cytochrome C but also on the percentage ratio between its native and partially denatured forms. The release of the (CytC-CL~) complex into the cytoplasm leads to activation of membrane lipid peroxidation processes and a cascade of enzymatic reactions leading to apoptosis. Parameters investigated by kinetics of activated chemiluminescence, based on the data of cancer cells, confirm the properties to resist apoptosis and mutations, i.e. the reason for the appearance of different kinds of metastasis. The results of modelling are applicable to the analysis of natural apoptogens, which are the basis for the creation of drugs and lead to increased resistance of the organism to the effects of various factors.

The use of methyl green dye in flow cytometry to monitor the viability of Corynebacterium glutamicum cells was studied. The use of methyl green has an important advantage—its spectra do not overlap with the fluorescence spectra of other fluorophores. Our study demonstrated that the fluorescence spectra of live cells of Corynebacterium do not overlap with the spectra of dead cells stained with methyl green, which is a significant benefit of such use in flow cytometry to monitor the viability of Corynebacterium glutamicum cells. It has been established that the optimal concentration of methyl green for staining C. glutamicum cells is in the range of 5–15 μg/mL. Under these conditions, methyl green is nontoxic to corynebacterial cells, which is confirmed by the coincidence of the titer (number) of living cells determined using flow cytometry or a standard microbiological method based on growth on the nutrient medium.

Abstract—Fructose–glucose syrups (FGSs) are widely used in the food industry as sweeteners. The use of inulo-containing raw materials, which include the Jerusalem artichoke, for their production leads to an increase in the proportion of fructose in the final product to 80–85%, which allows one to classify FGSs as a natural sweetener. Modern production of FGSs in the Russian Federation is based on the use of the enzyme exo-inulinase (EC 3.2.1.80), which hydrolyzes β-2,1-glycosidic bonds of the inulin molecule to produce fructose and glucose. In practice, an enzyme preparation based on this enzyme is added into fermenters of the extracted Jerusalem artichoke diffusion juice (DJ) before the microfiltration stage. However, in addition to the main polysaccharide, inulin (75–80%), DJ contains pectin (up to 5%), which creates an additional load on the microfiltration unit due to the viscosity of Jerusalem artichoke fermentolysates. Based on the fungal strain Penicillium verruculosum InuPel-10, a complex enzyme preparation (EP) of exo-inulinase/pectin lyase was obtained, which reduces the viscosity of Jerusalem artichoke extracts by 17–20%, while the yield of reducing sugars during hydrolysis using exo-inulinase/pectin lyase EP increased by 10%. The use of the complex EP InuPel-10 in the production of FGSs will reduce the cost of the EP by 10–16% due to the reduction in operating costs associated with a more fully loaded technological line for the production of this EP, in contrast to the production of individual enzymes and their further mixing in the required proportion.

Laboratory diagnostics of cholera and study of the properties of the pathogen Vibrio cholerae have not lost their relevance because of migration of populations and the possibility of importing this infection into our country. The emergence of post-genomic technologies opened a new era in the analysis of proteomes and enzyme systems of pathogens and their hosts during the infectious process. The rational use of these methods allows one to improve modern strategies for cholera express-diagnostics and evaluate the effectiveness of the disease treatment. Here, along with a retrospective analysis of the biochemical and genetic principles of the systematization of V. cholerae strains, new data on their metabolic features, which were obtained using MALDI-TOF, nanofluid, and chromatography-mass spectrometry in combination with 2D-electrophoresis and genetic methods, are presented. The structural features of O-antigens V. cholerae Ogava and Inaba and the mechanism of electron transfer along the respiratory chain are considered. The facts clarifying the role of protein kinases and the phosphorylation cascade in metabolic processes under the action of cholera toxin are presented. The inducing effect of the cholera toxin and hemolysin on IL-10-mediated inflammatory responses is described. New data on the role of the phosphoenolpyruvate system in glucose utilization and anaerobic glycolysis in the induction of cholera toxin are discussed. The introduction of the MALDI-TOF technology for biotyping and identification of microorganisms has led to the creation of a database of virtual collections for mass-spectrometric profiles of V. cholerae strains, and its use allows for identifying taxon-specific proteins and virulence markers of the bacterium. For this review, international and Russian databases were used.

Previously, it was demonstrated that recombinant spidroins rS1/9 and rS2/12 are able to glue various inorganic and organic materials. It is known that mussel foot proteins have unique adhesive properties associated with a high content of L-3,4-dihydroxyphenylalanine (DOPA), which is formed due to modification of tyrosine residues by the enzyme tyrosinase. We constructed a hybrid protein, which contains recombinant mussel foot protein 3 (Mfp3), as well as the recombinant tyrosinase used for modifying the tyrosine residues in DOPA in all recombinant proteins (rS1/9, rS2/12, and Mfp3 in the composition of the hybrid protein). Such modification led to an enhancement of the adhesive properties of these proteins, while the combination of modified spidroins with Mfp3-DOPA significantly increased their adhesive ability, which was demonstrated in the experiments on gluing various materials. The results obtained indicate the high potential of the hybrid protein containing recombinant modified Mfp3 for use alone or as an additive to recombinant spidroins for medical use.

The role of the microbiota in maintaining human health and the current state of research on the human microbiome, with a focus on the development and prospects for the use of modern methodological approaches, such as the “gut-on-a-chip” technology, are described in this review. The relationship between the microbiome and metabolomics is discussed, as are the main results of international projects on the human microbiome. The limitations of both cultural and purely genetic (metagenomics) approaches for studying the microbiota bank are highlighted. The central focus is on comparison of different gut models. Arguments in favor of a microfluidic biochip approach are presented. Finally, the regulatory aspects of the practical implementation of microarrays in drug development and personalized medicine are discussed briefly.

NAD⁺-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) plays a key role in the biofilm growth of this pathogen. Under these conditions, the reaction catalyzed by SauFDH is the main source of energy in the cell. Therefore, highly effective SauFDH inhibitors can be used as a basis for the development of fundamentally new drugs against S.aureus biofilms. The Nuclear Magnetic Resonance method is one of the most powerful and effective for studying the interaction of promising inhibitors with the enzyme globule. In this work, we optimized the cultivation conditions for the effective introduction of ²H, ¹³C and ¹⁵N isotopes into the enzyme, both individually and together. MALDI/TOF/TOF mass spectra of peptides of isotopically labeled enzyme preparations after tryptic hydrolysis of samples SauFDH ²H, SauFDH ²H,¹⁵N,¹²C and SauFDH ²H,¹⁵N,¹³C showed a uniform distribution of these isotopes in the protein molecule with a degree of enrichment of at least 95% in ²H and not less than 98% for ¹³C and ¹⁵N. This level of isotope enrichment is the maximum achievable in a research laboratory and is completely sufficient for NMR experiments.