Applied Biochemistry and Microbiology最新文献

The processes of biocatalytic synthesis of cefamandole and cefazoline, as well as four “chimeric” cephalosporins carrying functional groups of these antibiotics in the C3 or C7 position of β-lactam, were carried out using immobilized cephalosporin-acid synthetase under mild standard conditions. A higher efficiency of biocatalytic acylation of β-lactams with a 1(H)-tetrazolylacetic acid residue was demonstrated compared to acylation with mandelic acid residue. The chemical structure of the obtained compounds was confirmed using the HPLC-MS method. The possibility of using directly reaction mixtures for evaluating the antibacterial activity of the compounds synthesized without isolating the target products is demonstrated. The activity of the obtained cephalosporins against twelve microorganisms belonging to the genera Enterococcus, Acinetobacter, Serratia, Pseudomonas, Staphylococcus, and Escherichia was evaluated by the method of diffusion into agar. The activity of synthesized “chimeric” cephalosporins against four microorganisms was found: Escherichia coli VKPM B-6695, Staphylococcus aureus VKPM B-6646, Staphylococcus aureus VKPM B‑8171, and Staphylococcus epidermidis VKPM B-12635.

Nonylphenol is an aromatic organic compound that has an estrogen-like effect and has a negative effect on the human endocrine system. A method has been developed for the competitive determination of nonylphenol using magnetic particles, rabbit antiserum, nonylphenol conjugate with soybean trypsin inhibitor (STI) and biotin. The principle of the analysis is the formation of immune complexes on the surface of magnetite particles due to covalent immobilization of protein G through the oriented immobilization of polyclonal antibodies from rabbit serum during a competitive reaction between the free analyte (nonylphenol) and the bound one (as part of the nonylphenol-STI-biotin conjugate) for the binding sites of specific antibodies. The detection of formed immune complexes is proposed to be carried out using a streptavidin-polyperoxidase conjugate, which makes it possible to achieve a nine-fold gain in the level of the analytical signal. The developed ELISA using magnetite particles allows us to achieve a detection limit of nonylphenol at the level of 3.8 ng/mL, which is 14.5 times lower in comparison with the classic competitive ELISA (55 ng/mL). Based on the results of the experimental work, the optimized volume of the test sample was 500 μL, which makes it possible to concentrate low-contaminated samples by 17 times.

Using the previously engineered adipate-secreting Escherichia MG1655 lacI^Q, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, P_L-SD_φ10-atoB, P_trc-ideal-4-SD_φ10-fadB, ∆fadE, P_L-SD_φ10-tesB, ∆yciA, P_trc-ideal-4-SD_φ10-fabI, P_L-SD_φ10-paaJ, ∆aceBAK, ∆glcB as the core strain, the derivatives capable of enhanced synthesis of the target compound from glucose via the reversed fatty acid β-oxidation pathway were obtained. The respective effect was achieved due to the intensification of the tricarboxylic acid cycle in the cells. Prevention of multiple cycle turnovers, resulting from the inactivation of succinate dehydrogenase, had no pronounced effect on the formation of adipic acid by the recombinant. Upon intensification of the cycle due to enhancing anaplerotic oxaloacetic acetic acid formation from phosphoenolpyruvate, resulting from the increased expression of the native ppc gene, the synthesis of adipic acid increased by 1.2-fold up to ~390 μM. Enabling the formation of oxaloacetate from pyruvic acid, by introducing heterologous Bacillus subtilis pyruvate carboxylase in the cells , resulted in a 1.5-fold intensification of the cycle, concomitantly with the proportional increase in adipic acid secretion to ~496 μM. Subsequent inactivation of sdhAB genes in the strain increased the secretion of the target compound only slightly, and the adipic acid titer reached ~520 μM. The obtained data indicated a direct dependence of the efficiency of adipic acid synthesis by the engineered strains on the degree of intensification of the tricarboxylic acid cycle.

Methionine biosynthesis in most microorganisms proceeds in two alternative ways. Each pathway is catalyzed by independent enzymes and is tightly regulated by methionine. The transulfurylation pathway involves the formation of a cystathionine, and cysteine acts as a source of sulfur. The enzymes of this metabolic pathway are characterized in detail. The direct sulfhydrylation pathway involves the synthesis of homocysteine with the participation of an inorganic sulfur source directly from O-acetylhomoserine and is predominant in most classes of bacteria. The subject of this review is the properties and functioning of one of the least studied enzymes of the direct sulfhydrylation pathway—O-acetylhomoserine sulfhydrylase. A deep understanding of the mechanisms controlling the substrate and reaction specificity of O-acetylhomoserine sulfhydrylase is a necessary step in the rational redesign of the enzyme in order to create a promising catalyst for the synthesis of methionine and its derivatives, as well as, in combination with crystallographic data, for the development of new antimicrobial compounds based on effective enzyme inhibitors.

The reactivity during enzymatic hydrolysis of eight industrially produced samples of pulps and semichemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of the ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semichemical pulps obtained after green liquor cooking of hardwood. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical refining improves the performance of the enzymatic saccharification process.

The current information about hydrophobins, low molecular weight proteins synthesized by filamentous fungi, which are one of the strongest cellular biosurfactants, is summarized. The mechanism of biosynthesis of hydrophobins, the chemical structures and spectrum of its natural and synthetic isoforms, biological activity, and role in the regulation of vital processes of producers are presented. The potential for using hydrophobins in biotechnology has been demonstrated.

A method has been developed for producing analogs of fermented milk drinks from pumpkin seed meal, which is a massive waste product from oilseed production, using new strains of lactic acid bacteria (LAB) isolated from different samples of kumiss. Based on the results of screening 50 LAB isolates capable of fermenting milk and aqueous meal extracts in a wide pH range, three strains with the best growth characteristics were selected. These strains were identified as representatives of the genus Lacticaseibacillus, most closely related to L. rhamnosus and L. casei (with 99.93 and 99.65% similarity in 16S rRNA gene sequences). An optimal scheme for producing drinks has been selected, including grinding meal, optimized extraction with alkaline solutions, heat treatment of the extract to remove foreign microflora, introduction of the inoculum (3–5% vol/vol) of new LAB strains, and fermentation at 37^oС for ten hours. Compared with the fermented milk product obtained by fermenting milk with the same microorganisms, the drink made from meal extracts was distinguished by the absence of lactose and cholesterol, and increased content of unsaturated fatty acids (2.3 times) and protein (1.7 times), and the presence of essential amino acids in proteins. Thus, pumpkin seed meal, which is still used ineffectively, is a good basis for obtaining analogs of fermented milk products with beneficial properties. The developed method for producing lacto-fermented drinks can be adapted for processing other types of meals and cakes.

The results of studies of an intensive culture of a new species of bentoplanktonic diatom N. shiloi (Lee, Reimer et McEnery) Round, Hallsteinsen et Paasche 1999 isolated from the Black Sea are presented. Detailed descriptions are provided of the methods used to isolate the species into an algologically pure culture and its morphological and taxonomic features under light and electron scanning microscopy. The biochemical and production characteristics were also studied including the ability of the strain to accumulate fucoxanthin (Fx) and polyunsaturated fatty acids (PUFA) in laboratory conditions. During the exponential growth phase, the specific culture growth rate was µ = 0.8 1/day, and the maximum productivity P = 0.46 g dry weight/(L day). The accumulation of PUFAs in the biomass of N. shiloi reached 67.39 mg/g dry weight of algae. The Fx concentration in the biomass at the beginning of the stationary growth phase was 10 mg/g dry weight. The fairly high rate of Fx biosynthesis in microalgae cells, as well as the composition of fatty acids of the Black Sea strain, makes it possible to classify N. shiloi as a promising object in biotechnology.

A significant variation in the relative representation of copies of bacterial genes of dye-decolorizing DyP peroxidases typical for the genus Shewanella and a number of other microorganisms was found in the bottom sediments of freshwater reservoirs. It was found that the specific rate of decolorization of crystal violet in a laboratory bioelectrochemical system by a mixed culture of bottom sediments, which showed the highest representation of DyP genes, depended on the method of electrical stimulation of the external circuit and the concentration of the dye. After an increase in the concentration of more than 20 microns, the maximum speed was achieved in the presence of an ionistor polarly connected to the external electrical circuit of the bioelectrochemical system and amounted to 3.23 ± 0.11 μM/h, while with the opposite polarity connection, a minimum value of 2.07 ± 0.08 μM/h was observed. In the case of an open circuit and a resistor, similar indicators occurred with 2.88 ± 0.09 and 2.67 ± 0.12 μM/h, respectively. When analyzing the decolorization products, a consistent decrease in the maxima of the absorption bands of the dye was noted, indicating its more complete degradation by the mixed culture. The results may be of interest for the development of methods to improve the efficiency of bioelectrochemical methods of environmental biotechnology by electrostimulation of the external circuit.

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Te-nebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni²⁺-NTA agarose. The specificity constants (k_cat/K_M) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA, were 7, 4.2, and 0.9 (µM min)^–1, respectively. The optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable in the alkaline pH range, and in the presence of BSA, also in the acidic region. Peptidase was inhi-bited by synthetic inhibitors such as PMSF, TPCK, and chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e.g., stain removers, detergents, etc.).