NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

The effect of temperature on the exchange of di- and monovalent ions on polymethacrylic and polyacrylic cation exchangers in the range from 298 to 413 K is studied. It is shown that the differential enthalpy and the equilibrium coefficient simultaneously increase with temperature. The dependences of the enthalpy on temperature are linear. The effect of temperature on the enthalpy of ion exchange observed for all studied ion exchangers is much more strong than in the systems described in the literature with sulfonic acid cation exchangers and highly basic anion exchangers. The problem of taking into account the dependence of enthalpy on temperature when predicting the behavior of ion-exchange systems is discussed.

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD+ to NADP+ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.

Изучено влияние температуры на селективность и энтальпию обмена ионов Ni2+ − Na+ и Mg2+− Na+ на полиметакриловом катионите КБ-4П2 в интервале от 298 до 413 К. Показано, что при повышении температуры одновременно со значительным увеличением селективности к двухзарядному иону линейно увеличивается дифференциальная энтальпия. Наиболее сильно селективность возрастает в области «перегретых» растворов с температурой выше 373 К. Влияние ионного состава ионита на дифференциальную энтальпию значительно меньше, чем влияние температуры.

Formate dehydrogenases (FDHs) from different sources are systematically studied in our laboratory. Over the past few years, new genes of four FDHs from pathogenic bacterium Staphylococcus aureus (SauFDH), methylotrophic thermotolerant yeast Ogataea parapolymorpha (OpaFDH), yeast Saccharomyces cerevisiae (SceFDH), and moss Physcomitrella patens (PpaFDH) have been cloned and expressed in E. coli cells. By means of differential scanning calorimetry, a comparative study of thermal stability of new recombinant formate dehydrogenases and a number of FDHs from other sources has been performed. It was shown that two new enzymes, SauFDH and OpaFDH, are comparable to the FDH from Pseudomonas sp. 101 bacteria in their stability. SceFDH is the least stable FDH among the described formate dehydrogenases.

The problem of experimental determination of the differential enthalpy of ion exchange is considered. The determination of the enthalpy from experimental equilibrium coefficients is characterized by a high degree of error. A considerably more accurate method of determination of the differential enthalpy of ion exchange on selective ion exchangers is proposed. The method is based on the analysis only of the composition of the solution in the equilibrium system at two temperatures. The influence of temperature on the exchange of mono- and divalent ions on polyacrylic and polymethacrylic cation exchangers within the range from 273K to 400K is studied. It is shown than for all exchangers under study the differential enthalpy linearly increases with temperature. The role of the increase of ΔHn¯ with temperature in the process of a single-step dual-temperature separation is estimated. It is found that the increase of the upper temperature boundary leads to a very sharp increase of the degree of dual-temperature separation. This effect is most pronounced in superheated solutions. It is shown that when predicting changes in selectivity with temperature and the extent of purification of solutions of alkali metal salts from admixtures of divalent ions, it is necessary to take the temperature dependence of the ion exchange enthalpy into account.