Moscow University Chemistry Bulletin最新文献

To increase the frequency of homologous recombination (HR) during the transformation of the industrial Penicillium verruculosum 221-151 strain (VKM F-3972D), the ku70 gene encoding the Ku70 protein, which binds at sites of double-stranded DNA breaks and is involved in the repair process through nonhomologous end joining (NHEJ), is knocked out by the CRISPR/CAS9 method. Presumably, the new host strain, P. verruculosum ΔniaDΔku70, should have an increased frequency of homologous recombination during the transformation in comparison with the host strain P. verruculosum ΔniaD due to the integrative insertion of the expression cassette only through the HR mechanism. The pep1 gene encoding its homologous aspartate protease is chosen as a marker. However, it is shown that the knockout of the ku70 gene leads to a dramatic decrease in the frequency of cotransformation in the P. verruculosum ΔniaDΔku70 strain compared to the P. verruculosum ΔniaD strain at the same load of exogenous DNA (3 μg). At the same time, the number of copies of the pep1 gene in recombinant strains of the P. verruculosum Pep1 series (with the native Ku70) ranges from 3 to 28 copies, which indicates the predominance of the nonhomologous recombination mechanism (NHEJ).

The possibilities that open up when using instrumental methods such as voltammetry, surface-enhanced Raman spectroscopy (SERS), and mass spectrometry in tandem with enzymatic catalysis are considered. The basic principles of operation of electrochemical biosensors based on oxidases and dehydrogenases are described. It is shown that biosensors based on cyclic enzymatic reactions and substrate recycling have the best sensitivity. Variants of a significant improvement in the analytical capabilities of biosensor analysis due to the application of polymers for effective modification of the electrode surface and nondestructive immobilization of enzymes are considered. The data demonstrating how the use of enzyme labels expands the range of bioanalytical applications of SERS are presented. The possibility of a highly sensitive measurement of the activity of enzyme labels (peroxidase, alkaline phosphatase, β-galactosidase) by SERS, in fact, opens up a new universal platform for the development of protocols to determine various antigens. The main trends in the methodology development of proteomic studies by mass spectrometry and the role of proteases in the design of mass spectrometric experiments are considered on the example of the most commonly used proteases.

This review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present time. The results concerning the research on the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of multienzyme cellulase complexes; and the role of individual components of these complexes—basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase)—as well as their synergistic interaction, is described. The features of using reactors of various designs for bioconversion of plant raw materials are also described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, and reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physicochemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. The data on the creation of highly active strains of microscopic fungi—producers of cellulases and other carbohydrases using methods of induced mutagenesis—Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, and Chrysosporium (Myceliophtora) spp., as well as the data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them, are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EPs) that ensure highly efficient bioconversion processes of plant raw materials and create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EPs obtained using the P. verruculosum expression system are currently being produced at the Agroferment plant.

NAD(P)⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)⁺ to NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas sp.101 bacterium. Next, a sequence of six histidine residues (His-tag) was added to the N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus cells. These enzymes were purified, their kinetic parameters were determined, and thermal stability was studied. In the case of SoyFDH_L, which is similar to the natural form of the enzyme, both variants, with and without His-tag, the expression level is two times higher compared to the truncated variant. The addition of His-tag to the N-terminus of enzymes reduces the level of expression. Changing the sequence of the N-terminus, as well as introducing the His-tag sequence to the N-terminus, does not significantly affect thermal stability of the enzymes at temperatures of 50–56°C. However, due to the higher values of the activation enthalpy ΔH^≠ of the thermal inactivation process, the shortened form at normal temperatures is 3 times more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long version SoyFDH_L has lower values (K_{{text{M}}}^{{{text{HCOO}} - }}), and the short version SoyFDH_S has lower (K_{{text{M}}}^{{{text{NAD + }}}}) values. The introduction of His-tag into the N-terminus of enzymes does not affect their kinetic parameters.

This review considers the contribution of works carried out at the scientific school of Ilya Vasilievich Berezin to research on the kinetics and thermodynamics of penicillin acylase-catalyzed reactions. Methods for determining the activity of penicillin acylases, the reversibility of the enzymatic hydrolysis of a number of penicillins, cephalosporins, and related compounds, the influence of the β-lactam ring on the thermodynamics of the synthesis of new penicillins and cephalosporins by direct condensation as well as by acyl transfer, the issues of optimizing the conditions for enzymatic acyl transfer, and the use of supersaturated reagent solutions are discussed. The role of chromogenic substrates in the study of penicillin acylase, the possibility of using the methods of titration of active sites of the enzyme and the creation of “smart” biocatalysts based on penicillin acylase due to the formation of conjugates with stimulus-sensitive polymers are considered.

NAD(P)⁺-dependent formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the simplest reaction from chemical and biological points of view, oxidation of formate-ion to carbon dioxide coupled to NAD(P)⁺ reduction to yield NAD(P)H. Advances in the life sciences have shown that this reaction plays an extremely important role in a wide variety of organisms. The areas and types of practical applications of FDH are also permanently expanding. The review analyzes the key steps in the development of our knowledge on the role of formate dehydrogenase in living systems. Achievements in creation of highly efficient catalysts based on FDH for classic biotechnology as well as for new areas are also considered. The importance of a correct selection of the starting FDH form for the purpose of a biocatalyst design with required properties with minimal costs is demonstrated. The prospects for the use of FDH for CO₂ fixation of CO₂ are discussed.

Solubility profile of ketoconazole in the aqueous binary mixtures of ethylene glycol is measured by a shake-flask method at 293.2–313.2 K and mathematically represented by some cosolvency models (e.g. van’t Hoff, the λh equation, the Yalkowsky, the Jouyban–Acree, and the Jouyban–Acree–van’t Hoff models) and the models’ performances are illustrated by mean relative deviations. The density values of ketoconazole saturated solutions are also determined and represented by Jouyban–Acree model.

The nature of radical cations stabilized in irradiated frozen 1,3-butadiene diepoxide/CF₃CCl₃ solutions was determined via low-temperature UV/Vis spectroscopy, electron paramagnetic resonance spectroscopy, and quantum chemistry. It was found that the cyclic radical cations yielded as radiolysis products undergo C–C bond cleavage in both oxirane cycles under the action of light with further elimination of molecular formaldehyde resulting in distonic ^•CH₂OC⁺CH₂ radical cations.

Four amino phases representing 3-aminopropyl silica gel batches with different numbers of grafted functional groups are compared. To evaluate the chromatographic properties of the adsorbents, the Tanaka test for hydrophilic stationary phases is used and the retention of polar substances of various classes in the HILIC (hydrophilic) mode is studied. It is shown that even small changes in the nitrogen content between different batches of 3-aminopropyl silica gel have a significant effect on the retention of polar analytes. The hydrophilicity of a substrate is shown to have the greatest effect on its chromatographic behavior, and its evaluation using the Tanaka test is the basis for selecting a batch to separate specific classes of polar substances or for further modification to obtain new phases.