Moscow University Chemistry Bulletin最新文献

Our earlier annotation of the genome of the yeast Ogataea parapolymorpha DL-1 made it possible to identify five genes of potential D-amino acids oxidases. All opadaao1–opadaao5 genes were cloned and expressed in E. coli. Four OpaDAAO1-OpaDAAO4 enzymes were obtained in highly purified form and their catalytic properties were studied. It was found that among all DAAO described in the literature, the enzyme OpaDAAOl has the highest catalytic constant k_cat with D-Ala, which makes it promising for practical applications. However, in addition to good catalytic parameters, effective application of the enzyme in practice requires stability and knowledge of the inactivation mechanism, including at elevated temperatures. In this paper, we study the effect of elevated temperatures on the stability of OpaDAAOl. The enzyme is shown to have higher thermal stability than the majority of other D-amino acid oxidases. The kinetics of OpaDAAOl inactivation at different temperatures, at the initial concentrations of the enzyme, and in the presence of exogenous FAD are studied. A possible kinetic scheme of inactivation is proposed based on the data obtained.

E. coli strains are created as producers of recombinant β1-adrenoreceptor epitopes as part of chimeric proteins. The corresponding epitope sequences are located in the C-terminal region of the human heart fatty acid binding protein (hH-FABP) and separated from it by the linker sequence (Gly₄Ser)₃. A solid-phase enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies against the β1-adrenergic receptor in human blood serum based on a recombinant epitope is developed. Blood sera of patients (N = 76) with various diagnoses of cardiopathologies and other diseases are analyzed. A significantly high level of autoantibodies to the β1-adrenergic receptor is detected in some patients with a confirmed diagnosis of cardiovascular diseases, in most cases those with a diagnosis of acute myocardial infarction.

For human and chicken lysozyme, the relationship between changes in the parameters of enzyme adsorption on living Escherichia coli bacterial cells and the value of its effective bacteriolytic activity in the presence of glycine and charged amino acids is studied. It is shown for both human and chicken lysozyme that free amino acids added to a concentration of 1.5 mM for glycine or 5.0 mM for glutamate, aspartate, histidine, arginine, and lysine reduce the desorption constant of the enzyme on bacterial cells by factors of 1.4 to 2.0. At the same time, an increase in the bacteriolytic activity of lysozyme by factors of 1.5 to 1.9 is also observed. Thus, the enhancement of antibacterial activity in the presence of glycine and charged amino acids can be explained by the improvement in the productive sorption of the enzyme on the substrate of the bacterial cells.

D-amino acid transaminase from Aminobacterium colombiense was applied for (R)-selective amination of 2-oxobutyrate, 2-oxovalerate and 2-oxo-4-phenylbutyrate to produce non-natural D-amino acids—D-homoalanine, D-norvaline and D-homophenylalanine. To increase the product yield of D-amino acids, a one-pot three-enzyme system was developed. The system included transaminase from A. colombiense, (R)-2-hydroxyglutarate dehydrogenase and glucose dehydrogenase and effectively shifted the equilibrium of transamination reaction toward the products. The system functioned at both neutral and slightly alkaline pH. We found that at high substrate concentrations (500 mM) transaminase from A. colombiense was inhibited by the products accumulated in the system. The optimization of operational conditions of the three‑enzyme system led to the following yields of the target products: 435 mM D-homoalanine, 320 mM D-norvaline and 47.5 mM D-homophenylalanine; the enantiomeric excess of the produced D-amino acids exceeded 99.5%.

A technique for lateral flow hybridization analysis of specific bacterial beta-lactamase mRNAs, including the preparation of a biotinylated target DNA and its analysis on a test strip with immobilized oligonucleotide probes, is developed. When the target DNA and probe structures are complementary, DNA duplexes are formed in the test zone of the strip, which interact with the conjugate of streptavidin and gold nanoparticles. The technique is used to determine the transcripts of the genes encoding TEM-type beta-lactamases isolated from the culture of E. coli producing the corresponding recombinant beta-lactamase. It is shown that single-stranded 330 bp target DNA is determined most effectively in lateral flow analysis.

NAD⁺-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5–3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in (K_{{text{M}}}^{{{text{NA}}{{{text{D}}}^{{text{ + }}}}}}); the value of k_cat has not changed. When T250H is replaced, an increase in (K_{{text{M}}}^{{{text{NA}}{{{text{D}}}^{{text{ + }}}}}}) is observed, k_cat decreases from 20 to 13 s^–1. The replacement of K368H led to a slight increase in (K_{{text{M}}}^{{{text{NA}}{{{text{D}}}^{{text{ + }}}}}}), k_cat decreased from 20 to 6 s^–1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in (K_{{text{M}}}^{{{text{NA}}{{{text{D}}}^{{text{ + }}}}}}) and (K_{{text{M}}}^{{{text{HCO}}{{{text{O}}}^{ - }}}}). A bigger effect was observed for (K_{{text{M}}}^{{{text{NA}}{{{text{D}}}^{{text{ + }}}}}})—the difference was more than 10 times. For mutant SauFDH with insertions k_cat significantly reduced to 4 s^–1. Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH.

Low molecular weight RGD peptides and RGD mimetics are widely studied as ligands targeting the corresponding receptor in the diagnosis and therapy of cancer, as well as in the field of bone tissue regeneration. Some of them are undergoing preclinical trials. The aim of this study is to select the optimal variants of the ligand structure based on an aliphatic RGD mimetic. By methods of molecular modeling (blind docking and active site docking), the most advantageous constructions for the formation of a stable complex with the integrin α_Vβ₃ are determined. A scheme is developed and two lipotripeptides Gnd-GABA-Gly-Asp(C₁₆)₂ and Gnd-β-Ala-Gly-Asp(C₁₆)₂ with the potential ability to inhibit this receptor on the surface of tumor tissues are synthesized.

TEM experimental data for “Silver–Cholesterol” (Ag–Ch) and “Silver–Thiocholesterol” (Ag–TCh) systems showed the formation of silver nanoclusters of (6.0 ± 0.5) nm and (2.0 ± 0.5) nm in size correspondingly. Supramolecular organization of the systems due to donor-acceptor or electrostatic interactions with cholesteric ligands led to the formation of elongated helix aggregates with plasmonic absorbtion in visible and near-IR spectral regions. Using the DFT /B3LYP5 method the structures of biligand complexes of Ag₁₃ silver cluster with cholesteric ligands Ch and TCh were modeled and compared with the structure of monoligand complexes. O- and S-containing ligands were coordinated by vertices on different sides of the Ag₁₃ icosahedron. The energies of model reactions of the formation of monoligand and biligand complexes are compared. A tendency of the Ag₁₃ metal cluster to attach more ligands was found out. It can be assumed that the system retains the tendency of further aggregation with the attaching of the additional ligand molecules.

Cryoforming of gelatin systems with the antibacterial drug dioxidine is carried out. The study considers the effect of systems’ synthesis conditions (gelatin concentration in the precursor solution) on their structural characteristics, antibacterial activity, and drug release time. The composition and structure of the dioxidine/gelatin and dioxidine/hydrolyzed collagen systems are characterized by SEM, IR, and UV spectroscopy. The disk diffusion method is used to determine the antibacterial activity of the obtained systems against E. coli and S. aureus.

The possibility of identifying and authenticating cow milk powder by the method of chemical colorimetry of the fluorescence of samples using a smartphone camera and chemometric analysis is shown. A fundamental test device for direct colorimetric analysis of milk powder is proposed, excluding the stage of sample preparation. Fluorescence excitation is carried out with a portable source of monochromatic UV radiation (λ = 365 nm). The colorimetric parameters of milk powder fluorescence in RGB space are recorded using a smartphone camera. We use chemometric processing of the calculated analytical signal, which makes it possible to reduce the analysis time and visualize the studied data. The data array of the fluorescence colorimetric parameters (RGB) are evaluated by principal component analysis (PCA) and hierarchical clustering analysis (HCA) using the XLSTAT software.