Methods for targeting enzymes exhibiting anticancer properties, such as methionine gamma-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 - MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Data on the determining influence of Shewanella oneidensis MR-1 (gram−) and Bacillus subtilis 168 (gram+) bacterial strains used for the biosynthesis of CdS nanoparticles (NPs) on the quantitative and qualitative composition of the protein coating and functional characteristics of the nanomaterial such as the biocidal and photocatalytic activity are presented. The novelty of the study is quantitative assessment of the proteins adsorbed on the surface of CdS NPs/Shewanella and CdS NPs/Bacillus. It is shown that the protein content on the surface of CdS NPs/Shewanella and CdS NPs/Bacillus substantially differs and is 34 and 5% of the total weight of the nanoparticles, respectively. The fundamental difference in the qualitative composition of the “protein corona” of CdS NPs/Shewanella and CdS NPs/Bacillus is confirmed. The presence of nitrogen-containing organic compounds of protein nature on the surface of the nanoparticles is confirmed by Fourier-transform infrared (IR) spectroscopy. The possibility of using biogenic CdS NPs as biocidal agents of a new generation against a wide range of microorganisms as well as photonanocatalysts for the decoloration of synthetic dyes is proven. A difference in the level of functional activity of CdS NPs depending on the strain used for their biosynthesis is found and higher biocidal and photocatalytic activity of CdS NPs/Bacillus in comparison with CdS NPs/Shewanella is shown. The need for selecting bacterial strains to produce nanoparticles with high functionality is demonstrated.

Based on the Komagataella kurtzmanii yeast, a strain producing Tribolium castaneum recombinant procathepsin (rpTcCathL) has been obtained; this strain provided biosynthesis and secretion of at least 0.5 g/L of the target protein as a result of cultivation in flasks on a medium of complex composition. It was determined that the pH of the yeast culture medium (pH ≥ 6.5) is the key factor that influences the biosynthesis and accumulation of procathepsin. A new chromatographic purification scheme was developed, which allows one to obtain recombinant procathepsin with a yield of about 30

The development of recombinant modified derivatives of human glucose-dependent insulinotropic polypeptide (rmGIP) and glucagon-like peptide 1 (rmGLP-1) has been carried out as part of the project to create a prototype of a two-component drug. The aims were to increase the activity of GIP derivatives and obtain antagonists of GIP and GLP-1 receptors for selective neutralization of the activity of the corresponding components of a promising drug. For this purpose, well-known mutations were introduced into the structure of the basic human rmGIP(1‒42)h variant: a deletion of residues 32–42 and a H18R substitution, which is species specific for the mouse/rat hormones. The hypoglycemic activity of the drugs was measured using a glucose tolerance test on healthy mice. In most cases, the engineered mutations turned out to be unexpectedly ineffective or did not affect the hypoglycemic activity of GIP derivatives at all. The maximum two-fold increase in activity was recorded only in the modified rmGIP(1‒31) rat variant, which contained both mutations simultaneously. Inactivated rmGIP(3‒31)rat and rmGLP-1(3‒31) derivatives, containing the deletion of two N-terminal residues, specific for natural antagonists of the GIP and GLP-1 receptors (GIPR and GLP-1R, respectively) individually exhibited the expected dose-dependent antagonistic activity. At the same time, their equimolar mixture, instead of the expected additive effect, showed a complete loss of sugar-increasing activity. Based on the obtained results, we formulated the hypothesis about the ability of metabolites of the derivatives of incretin hormones GIP and GLP-1 to interact with each other in the process of glycemic regulation. This fact should be taken into account when studying the mechanisms of glycemic control and developing drugs based on agonists and antagonists of GIP and GLP-1 receptors.

The results of the microbial synthesis of extracellular silver nanoparticles (Ag NPs) in a liquid medium using the metal-reducing Shewanella oneidensis MR-1 strain and silver-nitrate salts are presented. The shape of the Ag NPs is determined by scanning transmission electron microscopy as close to spherical; more than 50% of Ag NPs have dimensions from 10 to 15 nm. The spectra of energy-dispersive X-ray microanalysis show the presence of Ag peaks in all samples of biogenic Ag NPs. The interplanar distances in the crystals of Ag NPs are 2.71 Å. Fourier-transform infrared (IR) spectroscopy confirms the presence of nitrogen-containing organic compounds of protein nature on the surface of the nanomaterial. The values of the hydrodynamic diameter, zeta-potential, and optical parameters of Ag NPs are determined by dynamic light scattering and spectrofluorimetry. It is shown that a large amount of Ag NPs is present on the surface of S. oneidensis MR-1 cells during biosynthesis. A technological approach is proposed to increase the yield of nanomaterial using the ultrasound treatment of cells, followed by the isolation of Ag NPs. It is shown that the compositions of the protein corona in Ag NPs obtained by the standard method and isolated from the cell surface after ultrasound treatment, as well as the size of Ag NPs, have some differences. A technological method for producing powdered preparations of nanomaterial by lyophilization (freeze drying) of aqueous suspensions of Ag NPs and cellular biomass containing Ag NPs is developed. The possibility of restoring preparations in the form of aqueous suspensions without the agglomeration and sedimentation of nanoparticles is shown. A high biocidal activity of all forms of the nanomaterial as antimicrobial agents of a wide spectrum of activity against gram-positive and gram-negative bacteria and microscopic fungi, including yeasts and phytopathogenic mold fungi, is established, which can be used to create polymer nanocomposites of varying nature with antibacterial properties.

-In this work, we report on the effect of Shewanella oneidensis MR-1 and Bacillus subtilis 168 bacteria used for the biosynthesis of CdS nanoparticles (CdS NPs) on the level of antimicrobial activity of CdS NPs/Shewanella and CdS NPs/Bacillus against a wide range of microorganisms: gram-positive, gram-negative bacteria, yeast and mold fungi. It is shown that S. oneidensis MR-1 and B. subtilis determine the quantitative characteristics of the nanomaterial, the content of protein and the CdS fraction in CdS NP samples. The use of the atomic emission spectroscopy method to determine the elemental content of cadmium and sulfur, as well as the densitometric analysis of electropherograms of protein adsorbed on the surface of biogenic CdS NPs demonstrate a significant difference in the ratio of these components in CdS NPs/Shewanella and CdS NPs/Bacillus. It is found that the proportion of CdS in CdS NPs/Bacillus exceeds by 1.4 times this parameter for CdS NPs/Shewanella. At the same time, the proportion of protein in CdS NPs/Bacillus is reduced compared to CdS NPs/Shewanella. It is shown that the level of antimicrobial activity of CdS NPs/Bacillus in relation to all test cultures studied exceeds the same parameter for CdS NPs/Shewanella. It is assumed that an increase in the proportion of CdS in CdS NPs/Bacillus may be the cause of a higher level of biocidal activity of CdS NPs due to an increased amount of released Cd2+ ions, which lead to the generation of reactive oxygen species and the destruction of microbial cells. Thus, the importance and necessity of selecting bacterial strains for the creation of functionally active nanoparticles with optimal characteristics is proved. The results of this research confirm the prospects of utilizing biogenic CdS NPs as targeted antimicrobial agents.

In addition to the previously developed recombinant modified human glucagon-like peptide 1 (rmGlp1, glypin), a recombinant modified human glucose-dependent insulinotropic peptide (rmGip) has been obtained. A new universal reverse-phase HPLC technique has been proposed that allows quantitative analysis of rmGlp1 and rmGip separately and as part of a two-component preparation. The design of recombinant human rmGip according to the protocols developed for glypin (the glypin formula) makes it possible to produce one-component and two-component preparations with various rmGip and rmGlp1 protein ratios ranging from 1 : 0 to 20 : 1, using cell biomass samples mixed in predetermined proportions. Studies of human rmGip activity in a mouse model revealed reduced specific activity and signs of weak antagonistic effects. In this regard, there is a need for further study of human rmGip activity in a mouse model, including the use of alternative mouse or rat rmGip.

Type 2 diabetes is one the most common metabolic diseases, which is obviously the price of lifestyle changes for many people. While being dangerous in itself, this disease provokes other metabolic disorders, such as obesity and neurodegenerative diseases such as Alzheimer’s disease. Pharmacologists are very active in creating drugs for these illnesses. The design of synthetic highly active and stable analogues of incretins, peptide hormones produced by neuroendocrine cells, is one of the most promising research areas. Glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide are the best known incretin hormones. Analogues of the first peptide have already found application in medical practice. The next step in the creation of drugs for diabetes was the development of polyagonists, which combine the properties of several different peptide hormones responsible for glucose homeostasis. The combination of the properties of incretins gives hope for a synergetic network effect. In the past few years, the creation of such co-agonists has progressed very rapidly. In some cases, the results of clinical trials have already been obtained; however, they often contradict each other. This difficult situation was the main motivation for writing the present review.

The study compared effectiveness of intranasal administration of glypin (human recombinant modified glucagon-like peptide-1) and reference drug Victoza in BALB/c mice. The minimum effective dose of intranasal glypin was 0.5 mg/kg, and a 2-fold elevation of this dose increased the parameters of glypin activity up to the maximal levels. During the first 2 h after intranasal administration, the effectiveness of glypin greatly surpassed that of Victoza. Duration of action and the time course of antihyperglycemic activity of intranasal glypin (1 mg/kg) matched to the best parameters attained during its subcutaneous application. A high effectiveness of intranasal glypin opens the vistas to its further examination and employment.

The double mutant of Wolinella succinogenes L-asparaginase (Was72) with the V23Q and K24T substitutions in the C-terminal region of the N-terminal loop enclosing the active site was crystallized in the apo form and in complexes with L-aspartic and L-glutamic acids. This mutant exhibits glutaminase activity eight times lower compared to the wild-type enzyme. Crystals of the apo enzyme were grown in two modifications (sp. gr. P 2 1 and sp. gr. P 22 1 2 1 ). Crystals grown in the presence of both aspartic and glutamic acids belong to the same space group ( P 2 1 ) but have different unit cell parameters. The X-ray diffraction data sets were collected from all types of crystals at 1.65–2.00 Å resolution. All X-ray diffraction data sets are suitable for the determination of the three-dimensional structure of the enzyme.

The isolation, heterologous gene expression, and characteristics of a new β-glucanase from Bacillus pumilus is described. The bgl1 gene from the Bacillus pumilus Bg57 VKPM В-13195 strain, which consists of 729 nucleotides, encodes a secreted endo-β-l,3(4)-D-glucanase (EC 3.2.1.6) containing 214 amino acids and 28 residues of the putative signal peptide in the N-terminal area. The nucleotide sequence of bgl1 and amino acid sequence of the mature Bgl protein have the highest homology (89 and 95%, respectively) with the sequences for β-l,3-l,4-glucanase from Bacillus licheniformis. A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. The purified recombinant enzyme of Bgl was active towards barley β-glucan and lichenin. With barley β-glucan, the optimal pH and temperature were shown to be 6.0 and 50°C, respectively; Km and Vmax were 2.2 mg/mL and 3036.4 μmol/(min mg), respectively. The recombinant protein Bgl showed a high specific activity, thermal stability, and resistance to digestive enzymes. Other characteristics of the recombinant protein, including pH stability and sensitivity to metal ions and chemical reagents, were also determined.

A simple and environmentally safe method for obtaining stable nanoparticles of metal sulfides nanoparticles – NpAg2S, NpCdS and NpZnS was developed using different strains of microorganisms in an aqueous solution of metal salts and sulfur sources at the Research Center "Kurchatov Institute" – GosNIIgenetika. The concentration of nanoparticles is 1–4 mg/ml in aqueous suspensions. Using of the methods of electron microscopy, spectrofluorimetry, dynamic light scattering determined the main characteristics of biogenic nanoparticles: shape, size distribution, crystal structure, effective diameter, luminescent spectrum, zeta potential. According to its characteristics, these nanoparticles are referred to quantum dots. It is established that the stability of nanoparticles in aqueous suspensions is due to protein molecules adsorbed on the surface of nanoparticles, which are supplied by cells of microorganisms. Effective immobilization of biogenic nanoparticles on the surface of various polymer supports has been carried out. Biogenic nanoparticles along with nanoparticles obtained by physico-chemical methods can be used as fluorophores for imaging of biological processes, also as photocatalysts, solar cells and for new nanocomposite materials.