Inhibition of β-lactamases involved in the development of bacterial resistance to β-lactam antibiotics is promising for restoring their effectiveness as antibacterial drugs. In this work, we screened the inhibitory activity against some serine β-lactamases of class A among humic substances of various origins, which represent complex organic matrices, as well as their narrow fractions obtained by separating preparations by acidity and polarity. The structural and group composition of broad fractions was characterized by 13C NMR spectroscopy; the molecular composition of narrow fractions was characterized by Fourier transform-ion cyclotron resonance mass spectrometry. The widest specificity of inhibition against various ESBL (TEM-12, TEM-17, TEM-18) and the inhibitor-resistant β-lactamase TEM-34 was possessed by the fraction of humic acids of brown coal CHA-I, isolated at pH 9: the values of residual activity of TEM -12, TEM-17, TEM-18, and TEM-34 were (

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.

A technique for lateral ow hybridization analysis of speci c bacterial beta-lactamase mRNAs has been developed, including the preparation of a biotinylated target DNA and its analysis on a test strip with immobilized oligonucleotide probes. When the DNA target and probe structures are complementary, DNA duplexes are formed in the test zone of the strip, which interact with the streptavidin conjugate with gold nanoparticles. The method was used to determine the transcripts of the TEM-type betalactamase genes isolated from the culture of E. coli - producers of the corresponding recombinant betalactamase. It has been shown that singlestranded DNA target of 330 bases was revealed most effectively in lateral ow analysis.

A biosensor based on the analyte-induced aggregation of functionalized nanoparticles recorded using two optical signals of different physical nature is developed. The hydrodynamic diameter of the conjugates, measured by the dynamic light scattering (DLS) method, and the counting rate of scattered light pulses, proportional to the elastic static scattering intensity are used together as the analytical signals. This improves the accuracy and reliability of the obtained results. Both signals are determined in one measurement on a compact DLS analyzer, specially designed as a detecting device for optical nano-biosensors. The proposed approach was used to detect Aspergillus galactomannan, a marker of an invasive fungal infection that poses a serious threat for the lungs. The results of the determination of galactomannan in the culture fluid of Aspergillus fungi, bronchoalveolar lavage (BAL) as well as in the calibration solutions, prepared on the basis of blood plasma and galactomannan calibrator are presented. A good correlation of the data obtained using nano- biosensor with the results of ELISA is shown, while the sensitivity of the analysis is not inferior to ELISA, and sample preparation is significantly simplified and accelerated by eliminating the washing stage. The detection limit for galactomannan in BAL samples was 3 ng/ml when detecting by the hydrodynamic diameter and 0.4 ng/ml when detecting by the counting rate of scattered light pulses. The total duration of sample preparation and measurements for a set of 10 samples does not exceed 100 min. The developed nano- biosensor can be used for the point of care diagnostics.

We optimize the procedure for synthesizing gold nanoshells for their use as a sensitive label in an immunochromatographic assay (ICA). An immunochromatographic test system is developed to visually determine the concentration of an early cardio marker, a heart-type fatty acid-binding protein (H-FABP). Visual detection yields the detection limit of the procedure with gold nanoshells to be more 50% lower than that of gold nanoparticles; it is 0.5 ng/mL for H-FABP.

For the experimental study of the role of the R65 residue in the stabilization of β-lactamases with the substitution of M182T, predicted by the analysis of the residue interaction networks (RINs), homogeneous preparations of the recombinant TEM-type β-lactamases with substitutions of R65L, M182A, and a combination of R65L and M182T mutations were obtained. The kinetic parameters of these enzymes were determined for penicillin, ceftazidime, cephalothin and CENTA. None of the investigated substitutions changed the substrate specificity of enzymes against β-lactam antibiotics. The substitution R65L leads to a decrease in thermal stability; the substitution of M182A and the combination of substitutions of R65L and M182T improve the thermal stability of β-lactamase in comparison with the wild-type enzyme TEM-1. Using differential scanning calorimetry, it was determined the enthalpy (ΔH, kJ/mol) and the denaturation temperature (Tmp, °C) for β-lactamases TEM-1, TEM(M182A), TEM-135(M182T), and TEM (R65L + M182T), which are, respectively, 554.0 and 50.8, 573.7 and 51.7, 654.4 and 55.9, and 647.4 and 51.9. The hypothesis of molecular mechanism, explaining the stabilizing role of M182T substitution in TEM type β‑lactamases, was supplemented by the effect of changing the conformation of the R65 residue and the appearance of its new contacts with the residues of the Ω-loop of β-lactamase.

An immunochromatographic test system for the combined analysis of two cardiomarkers, such as fatty acid binding protein (FABP) and troponin I (TnI), in serum and whole blood is developed. The main stages of the design of a multiparametric system are investigated, including the influence of the antibody position and gold nanoparticle size on the results. The detection limits of the method are found to be 10 ng/mL for FABP and 1 ng/mL for TnI, and the coefficient of variation did not exceed 10%. The developed test system allows us to confirm the diagnosis of acute myocardial infraction (N = 9). The analysis time is 15 min.

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2000 members: it includes genetically and functionally different bacterial enzymes capable to degrade the beta-lactam antibiotics. Beta-lactamases of molecular class A with serine residue in the active center are the most common ones. In the context of studies of the mechanisms underlying of evolution of the resistance, TEM type beta-lactamases are of particular interest due to their broad polymorphism. To date, more than 200 sequences of TEM type beta-lactamases have been described and more than 60 structures of different mutant forms of these enzymes have been presented in the Protein Data Bank. We have considered here the main structural features of the enzymes of this type with particular attention to the analysis of key mutations determining drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed a BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the TEM type beta-lactamases.

A gene-expression system has been designed to express the NDM-1 metallo-β-lactamase gene in E. coli cells. This system enables the synthesis of the recombinant protein in a soluble and active form. A method for the isolation and purification of the recombinant enzyme has been developed. The yield of the homogeneous protein preparation was 10–15 mg per liter of E. coli culture medium. The catalytic parameters of the recombinant NDM-1 β-lactamase were measured for ampicillin (K m = 185 μM and k cat = 585 s–1) and meropenem (K m = 85 μM and k cat = 160 s–1). These values correlate well with the literature data. The catalytic parameters for the chromogenic CENTA substrate (K m = 14 μM and k cat = 290 s–1) were obtained for the first time.

The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-β-lactam β-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial β-lactamase TEM-171, with a Ki of 88 μM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A β-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A β-lactamases. We also hypothesise that the presented route for finding non-β-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

A simple method for the rapid determination of prostate-specific antigen (PSA) in serum is reported using a lateral flow immunoassay with gold nanoparticles as the label. The method uses the intensity of colored test lines to determine PSA from 0.3 to 30ng/mL. The limit of detection was 0.3ng/mL and the coefficient of variation was less than 10%. The analysis time was approximately 20min. The novel method showed good correlation with enzyme linked immunosorbent assay (ELISA) measurements of prostate-specific antigen concentration in human serum with a linear regression coefficient of 0.985. The developed system was stable for at least 12 months when stored from +4 to 30 degrees C and has potential application for clinical practice.

Homogeneous preparations of recombinant β-lactamases TEM-1 and TEM-171 of molecular class A, differing by an amino acid substitution of valine at position 84 to isoleucine (Val84Ile), was obtained. The kinetic parameters of the β-lactamase TEM-171 were determined using a chromogenic substrate CENTA (K M eff = 23 μM, K cat = 102 s–1). The competitive inhibition of recombinant β-lactamases TEM-1 and TEM-171 by tazobactam was ascertained. The values of the inhibition constants in the hydrolysis of the CENTA substrate amount to 0.057 and 0.047 μM for TEM-1 and TEM-171, respectively. It was shown that the Val84Ile mutation leads to a decrease of TEM-171 enzyme thermal stability by 1.5 times.

Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

The article deals with prospects of using recombinant horseradish peroxidase in analytical biochemistry and biotechnology. Problems of recombinant horseradish peroxidase cloning in different expression systems, possible approaches to their solution, advantages of recombinant recombinant horseradish pe roxidase and recombinant horseradish peroxidase-fusion proteins for immunoassays are considered. Possibility for development of mediatorless bienzyme biosensor for peroxide and metabolites, yielding hydrogen peroxide during their transformations, based on co-adsorption of recombinant horseradish pe roxidase and the appropriate oxidase was demonstrated. The possibility to produce a fully active recombinant conjugate of recombinant horseradish peroxidase with human heart-type fatty acid binding protein, which may be used in competitive immunoassay for clinical diagnosis of acute myocardial infarction, and recombinant conjugates (Nand C-terminus) of recombinant horseradish peroxidase with Fab-fragments of the antibody against atrazine, which may be applied for atrazine pesticides detection, are demonstra ted for the first time.