Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices

AbstractNAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein α-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5–24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme

Thermal Decomposition of Co-Doped Calcium Tartrate and Use of the Products for Catalytic Chemical Vapor Deposition Synthesis of Carbon Nanotubes.

Thermal decomposition of Co-doped calcium tartrate in an inert atmosphere or air was studied using thermogravimetric analysis and X-ray absorption fine structure (XAFS) spectroscopy. It was shown that the powder substance containing 4 at.% of cobalt completely decomposes within 650-730 °C, depending on the environment, and the formation of Co clusters does not proceed before 470 °C. The products of decomposition were characterized by transmission electron microscopy, XAFS, and X-ray photoelectron spectroscopy. Surfaceoxidized Co metal nanoparticles as large as ∼5.6 ( 1.2 nm were found to form in an inert atmosphere, while the annealing in air led to a wide distribution of diameters of the nanoparticles, with the largest nanoparticles (30-50 nm) mainly present as a Co3O4 phase. It was found that the former nanoparticles catalyze the growth of CNTs from alcohol while a reducing atmosphere is required for activation of the latter nanoparticles. We propose the scheme of formation of CaO-supported catalyst from Co-doped tartrate, depending on the thermal decomposition conditions

Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae.

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+)

The Development of New Nanocomposite Polytetrafluoroethylene/Fe2O3 NPs to Prevent Bacterial Contamination in Meat Industry

The bacterial contamination of cutting boards and other equipment in the meat processing industry is one of the key reasons for reducing the shelf life and consumer properties of products. There are two ways to solve this problem. The first option is to create coatings with increased strength in order to prevent the formation of micro damages that are favorable for bacterial growth. The second possibility is to create materials with antimicrobial properties. The use of polytetrafluoroethylene (PTFE) coatings with the addition of metal oxide nanoparticles will allow to the achieving of both strength and bacteriostatic effects at the same time. In the present study, a new coating based on PTFE and Fe2O3 nanoparticles was developed. Fe2O3 nanoparticles were synthesized by laser ablation in water and transferred into acetone using the developed procedures. An acetone-based colloidal solution was mixed with a PTFE-based varnish. Composites with concentrations of Fe2O3 nanoparticles from 0.001–0.1% were synthesized. We studied the effect of the obtained material on the generation of ROS (hydrogen peroxide and hydroxyl radicals), 8-oxoguanine, and long-lived active forms of proteins. It was found that PTFE did not affect the generation of all the studied compounds, and the addition of Fe2O3 nanoparticles increased the generation of H2O2 and hydroxyl radicals by up to 6 and 7 times, respectively. The generation of 8-oxoguanine and long-lived reactive protein species in the presence of PTFE/Fe2O3 NPs at 0.1% increased by 2 and 3 times, respectively. The bacteriostatic and cytotoxic effects of the developed material were studied. PTFE with the addition of Fe2O3 nanoparticles, at a concentration of 0.001% or more, inhibited the growth of E. coli by 2–5 times compared to the control or PTFE without NPs. At the same time, PTFE, even with the addition of 0.1% Fe2O3 nanoparticles, did not significantly impact the survival of eukaryotic cells. It was assumed that the resulting composite material could be used to cover cutting boards and other polymeric surfaces in the meat processing industry

Composite Coating for the Food Industry Based on Fluoroplast and ZnO-NPs: Physical and Chemical Properties, Antibacterial and Antibiofilm Activity, Cytotoxicity

Bacterial contamination of meat products during its preparation at the enterprise is an important problem for the global food industry. Cutting boards are one of the main sources of infection. In order to solve this problem, the creation of mechanically stable coatings with antibacterial activity is one of the most promising strategies. For such a coating, we developed a composite material based on “liquid” Teflon and zinc oxide nanoparticles (ZnO-NPs). The nanoparticles obtained with laser ablation had a rod-like morphology, an average size of ~60 nm, and a ζ-potential of +30 mV. The polymer composite material was obtained by adding the ZnO-NPs to the polymer matrix at a concentration of 0.001–0.1% using the low-temperature technology developed by the research team. When applying a composite material to a surface with damage, the elimination of defects on a micrometer scale was observed. The effect of the composite material on the generation of reactive oxygen species (H2O2, •OH), 8-oxoguanine in DNA in vitro, and long-lived reactive protein species (LRPS) was evaluated. The composite coating increased the generation of all of the studied compounds by 50–200%. The effect depended on the concentration of added ZnO-NPs. The antibacterial and antibiofilm effects of the Teflon/ZnO NP coating against L. monocytogenes, S. aureus, P. aeruginosa, and S. typhimurium, as well as cytotoxicity against the primary culture of mouse fibroblasts, were studied. The conducted microbiological study showed that the fluoroplast/ZnO-NPs coating has a strong bacteriostatic effect against both Gram-positive and Gram-negative bacteria. In addition, the fluoroplast/ZnO-NPs composite material only showed potential cytotoxicity against primary mammalian cell culture at a concentration of 0.1%. Thus, a composite material has been obtained, the use of which may be promising for the creation of antibacterial coatings in the meat processing industry

Selenium Nanoparticles Can Influence the Immune Response Due to Interactions with Antibodies and Modulation of the Physiological State of Granulocytes

Currently, selenium nanoparticles (SeNPs) are considered potential immunomodulatory agents and as targets for activity modulation are granulocytes, which have the most abundant population of immune blood cells. The present study aims to evaluate the cytotoxic effect and its effect on the functional responses of granulocytes. In addition to the intrinsic activity of SeNPs, we studied the activity of the combination of SeNPs and IgG antibodies. Using laser ablation and fragmentation, we obtained nanoparticles with an average size of 100 nm and a rather narrow size evolution. The resulting nanoparticles do not show acute toxicity to primary cultures of fibroblasts and hepatocytes, epithelial-like cell line L-929 and granulocyte-like culture of HL-60 at a concentration of 109 NPs/mL. SeNPs at a concentration of 1010 NPs/mL reduced the viability of HL-60 cells by no more than 10% and did not affect the viability of the primary culture of mouse granulocytes, and did not have a genotoxic effect on progenitor cells. The addition of SeNPs can affect the production of reactive oxygen species (ROS) by mouse bone marrow granulocytes, modulate the proportion of granulocytes with calcium spikes and enhance fMLF-induced granulocytes degranulation. SeNPs can modulate the effect of IgG on the physiological responses of granulocytes. We studied the expression level of genes associated with inflammation and cell stress. SeNPs increase the expression of catalase, NF-κB, Xrcc5 and some others; antibodies enhance the effect of SeNPs, but IgG without SeNPs decreases the expression level of these genes. This fact can be explained by the interaction between SeNPs and IgG. It has been established that antibodies interact with SeNPs. We showed that antibodies bind to the surface of selenium nanoparticles and are present in aqueous solutions in a bound form from DLS methods, ultraviolet–visible spectroscopy, vibrational–rotational spectrometry, fluorescence spectrometry, and refractometry. At the same time, in a significant part of the antibodies, a partial change in the tertiary and secondary structure is observed. The data obtained will allow a better understanding of the principles of the interaction of immune cells with antibodies and SeNPs and, in the future, may serve to create a new generation of immunomodulators

Complexes of Ni^II, Co^II, Zn^II, and Cu^II with Promising Anti-Tuberculosis Drug: Solid-State Structures and DFT Calculations

Four new NiII, CoII, ZnII, and CuII complexes with the promising anti-tuberculosis drug (E/Z)-N′-((5-Hydroxy-3,4-bis(hydroxymethyl)-6-methylpyridin-2-yl)methylene)-isonicotino-hydrazide (LH) were synthesized and characterized by structural methods: single-crystal X-ray diffraction, vibrational spectroscopy, and mass spectrometry. The NiII, CoII, and ZnII metal ions form only amorphous phases with various morphologies according to mass spectrometry and IR spectroscopy. The CuII forms a crystalline 1D coordination polymer with the relative formula {[CuLCl]·0.5H2O}∞1. Even though the LH ligand in the crystalline state includes a mixture of E-/Z-isomers, only the tautomeric iminol E-/Z-form is coordinated by CuII in the crystal. The copper(II) complex crystallizes in the monoclinic P21/n space group with the corresponding cell parameters a = 16.3539(11) Å, b = 12.2647(6) Å, and c = 17.4916(10) Å; α = 90°, β = 108.431(7)°, and γ = 90°. DFT calculations showed that the Z-isomer of the LH ligand in solution has the lowest formation energy due to intramolecular hydrogen bonds. According to the quantum chemical calculations, the coordination environment of the CuII atom during the transfer of the molecule into the solution remains the same as in the crystal, except for the polymeric bond, namely, distorted trigonal bipyramidal. Some of the complexes investigated can be used as effective sensors in biosystems

Investigation of Aggregation and Disaggregation of Self-Assembling Nano-Sized Clusters Consisting of Individual Iron Oxide Nanoparticles upon Interaction with HEWL Protein Molecules

In this paper, iron oxide nanoparticles coated with trisodium citrate were obtained. Nanoparticles self-assembling stable clusters were ~10 and 50–80 nm in size, consisting of NPs 3 nm in size. The stability was controlled by using multi-angle dynamic light scattering and the zeta potential, which was −32 ± 2 mV. Clusters from TSC-IONPs can be destroyed when interacting with a hen egg-white lysozyme. After the destruction of the nanoparticles and proteins, aggregates are formed quickly, within 5–10 min. Their sizes depend on the concentration of the lysozyme and nanoparticles and can reach micron sizes. It is shown that individual protein molecules can be isolated from the formed aggregates under shaking. Such aggregation was observed by several methods: multi-angle dynamic light scattering, optical absorption, fluorescence spectroscopy, TEM, and optical microscopy. It is important to note that the concentrations of NPs at which the protein aggregation took place were also toxic to cells. There was a sharp decrease in the survival of mouse fibroblasts (Fe concentration ~75–100 μM), while the ratio of apoptotic to all dead cells increased. Additionally, at low concentrations of NPs, an increase in cell size was observed