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The thermal stability of protein enzymes is determined in vitro by measuring the enzymatic activity during incubation at constant temperature. Refolding of thermal inactivated enzymes is carried out both in vitro and in vivo, in the presence of chaperones, usually at temperature optimal for the particular enzyme for the manifestation of enzymatic activity. In the present work thermal stability of enzymes in vitro (using purified preparations) and in vivo (directly in the bacterial cell) has been determined. Bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi and Photorhabdus luminescens as protein substrates have been used. It is shown that the thermal stability of the P. luminescens and P. leiognathi luciferases in vivo in the Escherichia coli MG1655 dnaK^(+) and PK202 ΔdnaKJ14 strains is considerable higher than the thermal stability of "cell-free extract" luciferases. When an uncoupler of oxidative phosphorylation the carbonyl-cyanide-3-chlorophenylhydrazone (CCCP) that reduce the intracellular concentration of ATP to a minimum level, and the volatile hydrophobic substance (-)-Limonene (C10H16) as an inhibitor of chaperone-dependent refolding are added to the medium, the thermal stability of luciferases reduces almost to the level which is characteristic for the purified protein preparation. It is shown that the ATP-dependent chaperones ClpA and ClpB are essential for the increase of thermostability of luciferases in bacterial cells. Also, it is shown that the DnaKJE-dependent refolding of thermoinactivated luciferases is practically absent if the protonophore СССР or the hydrophobic substance (-)-Limonene was added to the bacterial suspension. Taking the data presented in this paper into account, it is necessary to consider the presence in bacterial cells of two different groups of ATP-dependent chaperones: 1st group (DnaKJE, GroEL/ES) is able to conduct the refolding both at low temperature after protein thermal inactivation and at high temperature at which protein thermal inactivation occurs; 2nd group (ClpA,ClpB, and possibly still unknown chaperones) is unable to conduct the standard refolding (i.e. at low temperature), but capable due to the hydrolysis energy of ATP of maintaining nonequilibrium stabilization of protein native forms at high temperature.

Enzymatic modification of meat with a high content of connective tissue is an effective mean, allowing to improve its properties and expand its use. Microbial enzymes have been extensively investigated as meat tenderizers. Compliance with safety requirements in terms of forecasting the development of various risks is essential for the use of these enzymes in food industry. The method of producing recombinant protease as a potential candidate for applications on meat tenderization was described in the article. The aim of this study was the production of recombinant Pichia pastoris with M9 peptidase gene from Aeromonas salmonicida. Material and methods. Objects: peptidase gene M9 (GenBank: CP000644.1 ASA_3723) Aeromonas salmonicida (strain of laboratory collection, isolated from the surface of raw meat), the vector plasmid pPic9K, competent E. coli DH5α cells, competent Pichia pastoris GS115 cells, culture fluid (QOL) from recombinant Pichia pastoris clones, beef shank samples. To obtain a recombinant strain, genetic engineering methods, the PCR method, and the bacteriological method were used. Polyacrylamide gel electrophoresis was used to separate and analyze the components of the supernatant. Enzyme activity was evaluated by HPLC-MS/MS using synthesized peptides. The impact of the supernatant from recombinant clones on the connective tissue of raw meat was assessed by histological method. Results and discussion. A metalloprotease M9 gene was cloned from the Aeromonas salmonicida (2748 bp) and expressed in Pichia pastoris. The molecular mass of the recombinant protein was estimated to be 120 kDa by SDS-PAGE. Histological analyses of the control and enzyme treated beef samples showed degradation intramuscular connective tissue, suggesting its effectiveness on meat tenderization. Conclusion. The recombinant strain Pichia pastoris, which produces the recombinant M9 peptide of Aeromonas salmonicida, has a specific enzymatic activity against collagen, the main component of the connective tissue of meat. The obtained recombinant peptidase M9 can be used as an enzyme softener of raw meat with a high content of connective tissue., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

The L1 protuberance of the ribosome includes two domain ribosomal protein L1 and three helices of 23S rRNA (H76, H77, and H78) with interconnecting loops A and B. Helix 78 consists of two parts, i.e., H78a and H78b. A comparison of the available structural data of L1-RNA complexes with the obtained kinetic data made it possible to determine the influence of the nonconserved regions of Thermus thermophilus L1-protuberance on the mutual affinity of the L1 protein and 23S rRNA. It has been shown that the N-terminal helix of the protein and 78b helix of 23S rRNA are essential for the formation of an additional intermolecular contact, which is separated in the protein from the main site of L1-rRNA interaction by a flexible connection. This results in a rise in the TthL1-rRNA affinity. At the same time, the elongation of the 76 helix has no effect on rRNA-protein binding.

Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.

Propionibacterium freudenreichii RVS-4-irf is a probiotic bacterium producing antimicrobial exometabolites applicable for foodstuff protection. Production of antimicrobial factors other than lowmolecular propionates was observed in media with and without trypton. A method was developed for production of the fraction of high-molecular mass exopolymers from the culture liquid. Their polypeptide nature was confirmed using proteinase K. The preparation of extracellular polypeptides from P. freudenreichii RVS-4-irf exhibited species-specific activity in suppression of fungal and bacterial growth.

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s–1; k cat/K m = 855 mM–1 s–1) has been 100–300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

In both prokaryotes and eukaryotes, the survival at temperatures considerably exceeding the optimum is supported by intense synthesis of the so-called heat shock proteins (HSPs), which act to overcome the adverse effects of heat stress. Among mycoplasmas (class Mollicutes), which have significantly reduced genomes, only some members of the Acholeplasmataceae family possess small HSPs of the α-crystallin type. Overproduction of a recombinant HSP IbpA (Hsp20) from the free-living mycoplasma Acholeplasma laidlawii was shown to increase the resistance of Escherichia coli to short-term heat shock. It has been long assumed that IbpA prevents protein aggregation and precipitation thereby increasing viability of E. coli cells. Several potential target proteins interacting with IbpA under heat stress were identified, including biosynthetic enzymes, enzymes of energy metabolism, and components of the protein synthesis machinery. Statistical analysis of physicochemical properties indicated that IbpA interaction partners significantly differ in molecular weight, charge, and isoelectric point from other members of the E. coli proteome. Upon shortterm exposure to increased temperature, IbpA was found to preferentially interact with high-molecular weight proteins having a pI of about 5.1, significantly lower than the typical values of E. coli proteins.

The accumulation of nanoparticles of colloidal silver with spherical shape in culture liquid of Azospirillum brasilense has been shown by transmission electron microscopy. Bacterial extracellular Mn-peroxidases were found to participate in silver reduction from silver nitrate with the formation of nanoparticles. A mechanism of extracellular biosynthesis of silver nanoparticles by A. brasilense bacteria was proposed

Synthesis of b-lactamases is one of the common mechanisms of bacterial resistance to b-lactam antibiotics including penicillins and cephalosporins. The widespread use of antibiotics results in appearance of numerous extended-spectrum b-lactamase variants or resistance to inhibitors. Mutations of 92 residues of TEM type were found. Several mutations are the key mutations that determine the extension of spectrum of substrates. However, roles of the most associated mutations, located far from active site, remain unknown. We have investigated the role of associated mutations in structure of b-lactamase TEM-72, which contain two key mutation (G238S, E240K) and two associated mutations (Q39K, M182T) by means of simulation of molecular dynamics. The key mutation lead to destabilization of the protein globule, characterized by increased mobility of amino acid residues at high temperature of modelling. Mutation M182T lead to stabilization protein, whereas mutation Q39K is destabilizing mutation. It seems that the last mutation serves for optimization of conformational mobility of b-lactamase and may influence on enzyme activity.

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K m) and maximum reaction velocities (V max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K i = 104.7 ± 21.7 mM, K m = 2.5 ± 0.5 mM, V max = 25.1 ± 1.5 U/mg) was observed.

Evolutionary connections were analyzed for endo-β-xylanases, which possess the GH10 family catalytic domains. A homology search yielded thrice as many proteins as are available from the Carbohydrate-Active Enzymes (CAZy) database. Lateral gene transfer was shown to play an important role in evolution of bacterial proteins of the family, especially in the phyla Acidobacteria, Cyanobacteria, Planctomycetes, Spirochaetes, and Verrucomicrobia. In the case of Verrucomicrobia, 23 lateral transfers from organisms of other phyla were detected. Evolutionary relationships were observed between the GH10 family domains and domains with the TIM-barrel tertiary structure from several other glycosidase families. The GH39 family of glycoside hydrolases showed the closest relationship. Unclassified homologs were grouped into 12 novel families of putative glycoside hydrolases (GHL51-GHL62).

Fluorescent proteins (FPs) are widely used as genetically encoded markers for noninvasive and quantitative study of biological processes. Development of biomarkers that fluoresce in the near-infrared spectral range allows the study of animals at a deeper level due to high permeability of tissues to light in this wavelength range, compared to the visible light. For widespread use of FPs, such properties as low molecular weight and the monomer become important. In this paper, we developed a FP called the GAF-FP and based on the chromophore- binding domain of bacterial phytochrome from Rhodopseudomonas palustris (RpBphP1). GAF-FP has a molecular mass of ~ 19 kDa, 2 times lower than that of other FP based on BphPs and 1.4 times less than the commonly used GFP-like proteins. Unlike most other near-infrared FP, GAF-FP is a monomer, has high photostability and its structure can withstand the introduction of small peptide inserts. Moreover, GAF-FP can covalently bind two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is found in mammalian tissues. GAF-FP with BV as a chromophore (GAF-FP—BV) has a main absorption band with a maximum at 635 nm and fluorescence maximum at 670 nm, whereby GAF-FP has a high signal to background ratio even if localized at a depth of several mm below the tissue surface. Apart from the near-infrared absorption band, GAF-FP—BV also has also an absorption band in the violet spectral range with a maximum at 378 nm. This property has been used by us to create a chimeric protein consisting of a modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We have shown that the chimeric protein is capable of resonance energy transfer from the substrate, which is oxidized by luciferase, to chromophore of GAF-FP—BV. In the absence of energy acceptor, RLuc8 catalyzes the cleavage of the substrate with light radiation having a peak of 400 nm. At the same time, as a part of GAF-FP—RLuc8 chimeric protein, the energy from the substrate is transferred to the chromophore of FP and then emitted in the near-infrared spectral range corresponding to GAF-FP fluorescence. These results open the way for the creation of new small near-infrared FPs based on various natural BphPs with a prospect of their wider use in cell and molecular biology.

Comparative characterization of the pigmented and nonpigmented Serratia marcescens strains and their extracellular nuclease mutants was carried out. Biomass accumulation by the mutant strains decreased on average by 20%, while proteolytic activity of the culture liquid was 4-5 times lower than in the case of the wild type strains. The mutants with impaired extracellular nuclease genes exhibited higher sensitivity to reactive oxygen species. Comparative analysis of motility of the strains revealed the highest flagellar activity in the wild type nonpigmented strain, while the cells of its mutant completely lost this feature.

The article considers a number of problematic issues concerning development of effective means of immune diagnostic of infectious diseases of bacterial and mycotic etiology related to approaches of choosing appropriate diagnostic targets.

Aim: Study the role of LPS in induction of anti-tularemia immunity in humans and animals., Materials and Methods: Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs)., Results: Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity., Conclusion: The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

The currently known methods of enzymatic β-lactam synthesis, as well as the enzymes and heterogeneous biocatalysts used for this purpose, are presented, and the published reports on advances in the field of enzymatic synthesis of selected antibiotics belonging to the groups of acidic penicillins and acidic cephalosporins are summarized in the present review. The key conditions and parameters of biocatalytic processes, such as the biocatalyst form, concentration of the precursor compounds, solvent type, pH, temperature, etc. are analyzed and compared, and guidelines for further optimization of β-lactam synthesis are given. The present review may be of use for a wide range of readers, as well as to enzymology and biotechnology experts.

Aim: Study protective activity of protein-containing antigens of pneumococcus, obtained from serotypes 6B, 10A, 14, 19F, 23F and 36R, against infection with heterologous strains of S. pneumoniae., Materials and Methods: S. pneumoniae strains of serotypes 3, 6B, 10A, 14, 19F, 23F and 36R, obtained from the collection of pneumococcus strains of Mechnikov RIVS, were used in the study. Protein-containing antigens of S. pneumoniae were isolated by acetone precipitations of supernatant fraction of culture medium. Protective activity of preparations of protein-containing antigens of pneumococcus as studied in experiments of active protection of BALb/c line mice., Results: The data obtained give evidence, that protein-containing antigens of pneumococcus, isolated from serotypes 6B, 10A, 14, 19F and 23F, effectively protect animals from subsequent infection with a heterologous S. pneumoniae strain of serotype 3 No. 11/56. Protection was noted at a level from 80 to 100% (p ≤ 0.05). Similar protective effect was detected in another experiment in a group of mice, immunized with preparations of protein-containing antigens of pneumococcus, obtained from serotypes 6B and 36R, against infection with a heterologous S. pneumoniae strain of serotype 3 No. 11/56. Protection was noted at a level of 90% (p ≤ 0.05)., Conclusion: The results of the experiments carried out allow to assume, that the main role in formation of cross-protection in experiments in animals is played by pneumococcus, proteins, that are a part of the studied preparations, and not polysaccharide antigens.

We report on theoretical efficiency of non-photochemical fluorescense quenching of phycobilisomes by the orange carotenoid protein. The created 3D computer model of the three-cylindrical phycobilisomes core allowed us to determine the distances between centers of mass of all phycobilin chromophores of the core and calculate the time and an average number of energy migration steps for the resulting non-radiative excitation transfer from the phycobilisomes to photosystem II. The obtained kinetic scheme equations for a way of energy transfer confirm the incomplete interception of energy flow in the phycobilisomes core by the orange carotenoid protein. Theoretical estimation of the rate of phycobilisomes quenching is in good agreement with experimental data.

Targeted delivery of semiconductor quantum dots (Q Ds) to tumors overexpressing HER2 cancer marker has been. demonstrated on immunocompromised mice bearing human breast cancer xenografts. To obtain targeted QDs complexes we applied the approach based on the use of protein adaptor system, RNAase barnase and its inhibitor barstar. Specific binding to target cancer marker was achieved through bivalent fusion protein containing two fragments of4D5scFv recombinant antibody and a fragment of barnase. QDs were conjugated to barstar, and final assembly of targeted complexes was obtained through non-covalent specific interaction of barstar, attached to QD, and barnase, that is part of the recombinant targeting protein. The efficient delivery of QDs to HER2-expressing tumor demonstrates the possibilities and prospects of the approach for targeted delivery of nanoparticles to cancer cells in vivo as the way to improve the efficiency of diagnosis and promote development of therapies based on the use of nanoparticles.

This mini-review summarizes recent data obtained in the process of creation of a versatile module platform suitable for construction of supramolecular theranostic agents. As an example, we consider multifunctional hybrid agents for imaging and elimination of cancer cells. The use of an adapter protein system barnase:barstar for producing targeted multifunctional hybrid structures on the basis of highly specific peptides and mini-antibodies as addressing modules and recombinant proteins and/or nanoparticles of different nature (quantum dots, nanogold, magnetic nanoparticles, nanodiamonds, upconverting nanophosphores, polymer nanoparticles) as agents visualizing and damaging cancer cells is described. New perspectives for creation of selective and highly effective compounds for theranostics and personified medicine are contemplated.

A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward's reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm-1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5-8-fold higher catalytic efficiency (kcat/Km) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.

Size exclusion chromatography was used to assess the relative size of intact and diphenylamine-treated (DPA, with suppressed carotenoid synthesis) peripheral light-harvesting complexes (LH2 complexes) of the sulfurbacterium Allochromatium minutissimum. Both LH2 complexes were nonamers and had the same elution volume V(e), coinciding with that for the LH2 complex of Rhodoblastus acidophilus (strain 10050). Their molecular mass was 150 kDa. Bot pheophytinization of bacteriochlorophyll (BChl) at low pH and treatment with the detergent LDAO, affecting the hydrophobic interactions between the neighboring protomers, result in the fragmentation of the ring of the isolated LH2 complexes and formation of 55-kDa fragments with molecular masses corresponding to one-third of the initial value. Fragmentation caused by both pheophytinization and detergent treatment was much more rapid in DPA-treated LH2 complexes than in the intact ones. The 55-kDa fragments formed at low pH values contained monomeric bacteriopheophytin, while the fragments of a similar molecular mass formed at pH 8.0 in the presence of the detergent contained monomeric BChl. The observed fragmentation was hypothesized to reflect the inherent C3 symmetry of the LH2 complexes, with the preliminarily assembled trimers used as building blocks.

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

The genome of Melioribacter roseus, one of two members of the recently described phylum Ignavibacteriae, was searched for the genes encoding proteins associated with copper transport or containing copper as cofactors, and the effect of Cu2+ concentration in the medium on M. roseus growth was investigated. Genomic analysis revealed a variety of copper-containing oxidoreductases in this facultative anaerobe. Three ATPases responsible for copper transport were identified. One of them (MROS_1511) was.probably involved in assembly of the copper-containing cytochrome c oxidase, while two others (MROS_0327 and MROS_0791) probably carried out a detoxification function. The presence of several copper-containing oxidoreductases and copper homeostasis systems in M. roseus is in agreement with the previously hypothesized origin of the phylum Ignavibacteriae from an aerobic ancestor common with those of Bacteroidetes and Chlorobi.

This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

Binase--Bacillus pumilus RNase--endonuclease cleaves the phosphodiester bond between the 3'-guanylic residue and the 5'-OH residue of adjacent nucleotides with the formation of corresponding intermediate 2',3'-cGMP. Subsequent hydrolysis of 2',3'-cGMP into 3'-phosphate is highly specific and occurs slowly So the question arises about the existing time of that positional isomer during RNA catalytic cleavage by binase and about 2',3'-cGMP role in antitumor activity of the enzyme: In present work by means of enzyme-linked immunosorbent assay we established that during catalytic cleavage of RNA by binase 2',3'-cGMP is preserved in reaction mixture for an hour, at the same time phosphodiesterases activation doesn't lead to the total elimination of 2',3'-cGMP. The highest amount of 2',3'-cGMP was observed under the pH 8.5, it reaches nanomolar concentration at initial RNA concentration of 100-1000 μg/mL. Exogenous 2',3'-cGMP, like its positional isomer 3',5'-cGMP, doesn't trigger an apoptosis of human lung adenocarcinoma A549 cells, which are sensitive to binase apoptogenic action. Taking into account data about binase internalization and activation of mitochondrial pores opening by 2',3'-cyclic guanosine phosphates we may consider that 2',3'-cGMP can contribute to the apoptosis initiated by binase only when 2',3'-cGMP is generated intracellularly.

For a long time the presence of knots in a protein structure was not admitted. However, the existence of proteins with various types of knots has now been proven. The functional significance of knotted topology remains unclear despite numerous assumptions. Studing the structure of knots in proteins and their impact on the acquisition of native structure of proteins is important for the understanding of protein folding as a whole. We review the types of knots in the proteins discovered to date, including trefoil knot, figure-of-eight knot, and more complex knots with 5 and 6 crossings of polypeptide chain. We survey the folding of knotted proteins as well.

P1-[11-(Anthracen-9-ylmethoxy)undecyl]-P2-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate, a fluorescent derivative of undecyl diphosphate 2-acetamido-2-deoxyglucose, was chemically synthesized. The ability of the compound to serve as acceptor substrate of D-rhamnose residue in the enzymatic reaction catalyzed by D-rhamnosyltransferase from Pseudomonas aeruginosa PAO1 was demonstrated.

Heat shock caused a more active formation of the "dormant" forms (minibodies), as well as increased production of extracellular membrane vesicles by Acholeplasma laidlawii PG-8A cells. Raise of the amount of the minibodies that have increased resistance to biogenic and abiogenic stress factors and pathogenicity may lead to more successful persistence of mycoplasmas in their hosts. Increased production of the extracellular membrane vesicles containing virulence factors by Acholeplasma laidlawii cells during stress may be an additional burden for the infected organism. It has been recently revealed that the vesicles of A. laidlawii contain appreciable quantities of small heat shock protein IbpA (Hsp20). In this paper, using immune-electron microscopy, have shown that at elevated temperature IbpA is associated with A. laidlawii minibodies. Perhaps, IbpA contributes to increased resistance and pathogenicity of the minibodies, keeping their proteins and polypeptides, including protein virulence factors in the folding-competent state.

Methionine γ-lyase [EC 4.4.1.11] participates in a methionine catabolism at a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. Lack of this enzyme at mammals allows consider it as a perspective target for rational antibacterial drug design. Currently in medical practice there are no the preparations based on an inhibition of methionine γ-lyase activity. We present results of the search of potential inhibitors of the enzyme using the NMR screening techniques based on identification of compounds, which able to bind specifically to their biological target. Study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds, capable to interact with enzyme. Identification of binding was carried out by means of saturation transfer difference (STD) spectroscopy and WaterLOGSY technique. At the final stage the experimental assessment of inhibiting ability of the selected compounds in the reaction of γ-elimination of L-methionine catalyzed by methionine γ-lyase was carried out. Binding constants of two leading compounds were determined using the WaterLOGSY method. The research expands structural group of potential inhibitors of methionine γ-lyase and allows approach to the design of the inhibitors with higher efficacy.

A diagnostic reagent application based on carbon nanoparticles covalently functionalized by streptococcus G-protein was investigated in order to construct a system for the monitoring and optimization of the affinity purification technology of rabbit polyclonal antibodies to alpha-fetoprotein. The developed system allows semiquantitative assessment ofthe immunoglobulin content (IgG) of most higher animals and human beings in blood serum samples and eluate in a short time (45 min). IgG detection sensitivity using the carbon-G-protein diagnosticum was 80 ng/mL. Approaches to stabilizing the components of the analysis system, which ensures the preservation of their functional properties during long storage, were developed. The storage life of the diagnosticum was more than 20 years, and that of immunosorbents was more than year and a half. A technique of long-term immunosorbent storage, used in the analysis, was developed. Application of the test-system is possible without the presence of registration equipment.

The study of sampling of strains of Y. pestis of main and altaic subspecies was implemented. The modern technique of identification of microorganisms was applied using MALDI-TOF mass spectrometry analysis. The evaluation of biological safety of method of sampling preparation was implemented. To supplement the identification base "BioTyper" the spectrum of typical strains of Y. pestis were obtained. The enhanced identification base was used to evaluate possibilities of application of MALDI-TOF technology for identification and taxonomic differentiation of Y. pestis from other representatives of genus of Yersinia. In the process of study a complete concordance of results of mass spectrometry identification and classic cultural method was observed. On the basis of mass spectrometry characteristic of analyzed sampling the differentiation between strains of Y. pestis of subspecies pestis and strains of subspecies altaica was implemented. The study results testify the effectiveness of application of mass spectrometry analysis for reliable interspecies and intraspecific differentiation of plague agent. The simplicity and velocity of sampling preparation and implementation of analysis and low cost of active storage allow considering the MALDI-TOF technology of mass spectrometry identification as highly perspective method for laboratory diagnostic of plague agent.

The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

The influence of cobalt (II, III) coordinative compounds with derivatives of dithiocarbamic acid on Bacillus thuringiensis IMV B-7324 peptidases with elastase and fibrinolytic activity and Eupenicillium erubescens and Cryptococcus albidus alpha-L-rhamnosidases have been studied. Tested coordinative compounds of cobalt (II, III) on the basis of their composition and structure are presented by 6 groups: 1) tetrachlorocobaltates (II) of 3,6-di(R,R')-iminio-1,2,4,5-tetratiane--(RR')2Ditt[CoCl4]; 2) tetrabromocobaltates (II) of 3,6-di(R,R')-iminio-1,2,4,5-tetratiane--(RR')2Ditt[CoBr4]; 3) isothiocyanates of tetra((R,R')-dithiocarbamatoisothiocyanate)cobalt (II)--[Co(RR'Ditc)4](NCS)2]; 4) dithiocarbamates of cobalt (II)--[Co(S2CNRR')2]; 5) dithiocarbamates of cobalt (III)--[Co(S2CNRR')3]; 6) molecular complexes of dithiocarbamates of cobalt (III) with iodine--[Co(S2CNRR')3] x 2I(2). These groups (1-6) are combined by the presence of the same complexing agent (cobalt) and a fragment S2CNRR' in their molecules. Investigated complexes differ by a charge of intrinsic coordination sphere: anionic (1-2), cationic (3) and neutral (4-6). The nature of substituents at nitrogen atoms varies in each group of complexes. It is stated that the studied coordination compounds render both activating and inhibiting effect on enzyme activity, depending on composition, structure, charge of complex, coordination number of complex former and also on the enzyme and strain producer. Maximum effect is achieved by activating of peptidases B. thuringiensis IMV B-7324 with elastase and fibrinolytic activity. So, in order to improve the catalytic properties of peptidase 1, depending on the type of exhibited activity, it is possible to recommend the following compounds: for elastase--coordinately nonsaturated complexes of cobalt (II) (1-4) containing short aliphatic or alicyclic substituents at atoms of nitrogen and increasing activity by 17-100% at an average; for fibrinolytic--neutral dithiocarbamates of cobalt (II, III) (4-5) (by 29-199%). For increasing the fibrinolytic activity of peptidase it is better to use dibenzyl- or ethylphenyldithiocarbamates of cobalt (III), which increase activity by 15-40% at an average. The same complexes, and also compound {(CH2)6}2Ditt[CoCl4] make an activating impact on alpha-L-rhamnosidase C. albidus (by 10-20%).

PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples.

Two families of specific inhibitors of type C lysozyme (Ivy and PliC) secreted from the periplasmic space are known in enterobacteria. Microbial capacity for distant lysozyme inactivation (antilysozyme activity) is most pronounced in the strains and species carrying homologues of the pliCgene. The pliC homologue localized in a -200-kbp megaplasmid of Klebsiella pneumoniae was shown to differ significantly in the amino acid composition of the coded polypeptide. Similar to the Salmonella enterica pliC homologue, it possesses a detachable signal part and contains identical functionally critical amino acids of the active center of the inhibitor. Antilysozyme activity of the pliC-positive K. pneumoniae strains was observed at the level corresponding to the highest values found inpliC-positive S. enterica. High level of the antilysozyme activity in K. pneumoniae strains containing the plasmid pliC homologue was found in all studied strains, unlike S. enterica strains carrying the known chromosomal pliC homologue.

CtXynGH30 is a carbohydrate active modular enzyme and component of cellulosome of Clostridium thermocellum. The full length CtXynGH30 contains an N-terminal catalytic module named as Xyn30A and a family 6 carbohydrate binding module (CBM6) at C-terminuis. Xyn30A was modeled by computer program Modeller9v8 taking crystal structure of XynC from B. subtilis as a template to generate the molecular model. Model refinement was done using energy minimization by implementing steepest descent algorithm with GROMOS96 43al force field. Quality assessment by Ramachandran plot showed that 91% amino acids lie in most favourable region and 9% in additional allowed region. Structural analysis depicted that Xyn30A has a (beta/alpha)8 barrel fold. Ad- ditionally, it had a beta-strand rich structure called 'side beta-structure' attached with main catalytic core. Structural superimposition reflected that Glu136 act as a catalytic acid/base while Glu225 act as a catalytic nucleophile. Multiple sequence alignment showed that these catalytic residues are conserved within the family. The docking results showed that these residues display polar interaction with linear and substituted xylo-oligosaccharides. The binding interaction of ligands depicted that aromatic amino acids Trp81, Tyr139, Trp143, Phe172, His198, Tyr200, Tyr227, Trp264 and Tyr265 create binding site pocket around the active site. We report overall structural feature, conserved active site residues and enzymeligand docking of first glucuronoxylan-xylanohydrolase (Xyn30A) of family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum.

The recombinant Escherichia coli strain pET35b-ARG, which overexpresses arginase I fused to a cellulose-binding domain (CBD), was developed. After preparing cellulose microspheres, arginase I was immobilized via the CBD of the fusion protein. Under optimal reaction conditions (40 degrees C, pH 9.5, 1 mM of Mn2+, 30 microl/ml of immobilized enzyme, 30 g/l of L-Arg, and for I h), the conversion rate of L-Arg was 98.7%. After 7 reuses of 30 microl of immobilized enzyme in 1 ml of catalytic solution, 153 mg of L-Orn with 97.3% purity was obtained. This indicated that the immobilization method was effective, feasible and could be used for the industrial production of L-Orn in the future.

The structural organization of inulinases from yeasts, fungi, and plants are researched. For studying their sizes, molecular weight, and permolecular organization, an approach consisting of a combination of atomic force microscopy with methods of dynamic light scattering, gel chromatography, and electrophoresis was used. It is shown that inulinases from Kluyveromyces marxianus and Aspergillus niger form geterodimers and inulinases from tubers of Helianthus tuberosus are present as both dimers and monomers. The role of various forms in the functional activity of inulinase molecules is discussed.

Based on Escherichia coli, highly sensitive specific lux-biosensors for the detection of tetracycline and beta-lactam antibiotics, quinolones, and aminoglycosides have been obtained. To make biosensors, bacteria were used that contained fungal plasmids pTetA'::lux, pAmpC'::lux, pColD'::lux, and plbpA'::lux, in which transcription of the reporter Photorhabdus luminescens luxCDABE genes occurred from the inducible promoters of the tetA, ampC, cda, and ibpA genes, respectively. The main parameters (threshold sensitivity and response time) of lux-biosensors were measured. The high specificity of biosensors responding only to antibiotics of a certain type was demonstrated.

Recombinant E. coli strains expressing the Bacillus cereus ATCC 14579T resD and resEgenes fused with the ubiquitin gene were constructed, and purification of the ResD and ResE proteins was performed. The approach used in the study allowed us to increase the protein yield of the electrophoretic homogeneous ResD andResE proteins without denaturation steps up to 150 mg per gram of wet cell weight.

The physicochemical and enzymatic properties of hybrid analogues of the Brevundimonas diminuta Gl7ACA-acylase (BrdGIA), containing the N-terminal chitin-binding domain of the bacterial chitinase (BrdG1A/NmChBD) or the C-terminal oligohistidine sequence (BrdGIA/H), were studied. An enhanced thermostability level of BrdG1A/NmChBD could suggest the stabilizing effect of the chitin-binding domain. An analysis of pH profiles of the enzymatic activity of recombinat BrdGIA analogues did not reveal significant differences: the catalytic activity of both variants changed slightly in the.interval ofpH values from 6.0 to 9.0 but drastically decreased at lower pH values. Both analogues demonstrated similar sensitivity towards denaturing agents: addition of 2.0 M ofguanidine chloride resulted in the complete inactivation of both enzymes. A scheme was developed for obtaining isolated recombinant alpha- and beta-subunits of BrdGLA. In vitro enzyme reconstructions indicated that the alpha-subunit was necessary for the formation of a correct spatial structure of the beta-subunit and for the formation of a functionally active enzyme.

The properties of amperometric biosensors based on methanol dehydrogenase (MDH), Methylobacterium nodulans cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition ofhydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135-0.5 and 0.032-1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 microM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.

Aim: Study system of activation of plasminogen in Vibrio cholerae., Materials and Methods: 75 strains of V. cholerae of various origins were used in the study. Plasminogen was isolated from human plasma by using affinity chromatography on L-lysine sepharose, alpha-enolase activity was determined by a direct method assuming transformation of 2-phosphoglycerate into phopshoenolpyruvate. Vibrios were destroyed by ultrasound disintegrator to isolate membrane Omp protein, intact cells were discarded by centrifugation and cell lysate was centrifugated for 1 hour at 105000 g. The precipitate was solubilized in buffer with 1% triton X-100 and passed through a column with DE-52 cellulose., Results: Vibrio cholerae O1 and O139 strains isolated from clinical specimens and water samples from open water bodies had the ability to bind by using alpha-enolase and transform human plasminogen into plasmin under the effect of outer membrane protein OmpT A protein with molecular weight around 40 kDa had proteolytic activity with a wide specter of substrate specificity, degraded fibrin, gelatin, collagen, protamine and activated plasminogen. Computer analysis showed that OmpT protein of cholera vibrion had a low degree of relation with Enterobacteriaceae omptins., Conclusion: The study carried out showed that vibrios have a system of activation of plasminogen that includes at least alpha-enolase and OmpT membrane protein. OmpT protein is assumed to belong to a new class of porins of Vibrionaceae family and its enzymatic activity may play a significant role in pathogenesis of infection.

The thermostable β-glucosidase A (TmBglA) from Thermotoga maritime is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (MI-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum tem- perature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that' these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G 174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.

With a number of experimental methods (circular dichroism, viscosity, intrinsic fluorescence and fluorescence labelling) the conformational folding-unfolding transitions in a compact monomeric form of the Caf1(13-149) subunit were studied under the action of guanidine hydrochloride in the temperature range from 5 to 45 degrees C. It has been shown that transitions always occur between two major states (unfolded and compact). It has made it possible to determine all main thermodynamic functions that characterize the compact state of the Caf1(13-149) subunit: temperature stability T(m), the free energy of stabilization deltaG(st), enthalpy deltaH(tr) and heat capacity jump deltaC collapse of the structure. Data obtained have been confirmed by an independent experiment on melting of fluorescently labeled proteins.

Atomic force microscopy with two types of probes - standard (radius of curvature R approximately 10 nm) and supersharp (R approximately 2 nm)- was used to determine CYP102A1oligomeric state. CYP102A1 images were obtained in a liquid, air and vacuum environment using the standard probes, also a ratio of monomers to oligomers (alpha) of CYP102A1 were determined as alpha=0.48:0.52. At the same time use of standard probes did not allow to resolve the structure of these oligomers. Supersharp probes allowed to obtain the data about the monomers to oligomers ratio, and also about the dimers/trimers/tetramers ratio in air and vacuum. So, a ratio alpha of CYP102A1 in liquid can be determined by the standard probes, and an oligomeric state of protein can be specified by the supersharp probes.