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Applied Biochemistry and Microbiology (v.47, #2)
Biosynthesis of arachidonic acid by micromycetes (review) by E. G. Dedyukhina; T. I. Chistyakova; M. B. Vainshtein (pp. 109-117).
Arachidonic acid (ARA, 5,8,1l,14-cis-eicosatetraenoic acid) is widely used in medicine, pharmaceutics, cosmetics, dietary nutrition, agriculture, and other fields. Microbiological production of ARA is of increased interest since the natural sources (pig liver, adrenal glands, and egg-yolk) cannot satisfy its growing requirements. Mechanisms for ARA biosynthesis as well as the regulation of enzymes involved in this process are considered. Review summarizes literature data concerning individual stages of microbiological ARA production, methods for screening of active strains-producers, physiological regulation of ARA synthesis in micromycetes (the effect of growth phase, medium composition, pH, temperature, and aeration), and effective technologies of fermentation and the product recovery. Information on the whole biotechnological process from strain selection to the ARA yield improvement and purification of the end product is presented.
MALDI-TOF mass spectrometric identification of novel intercellular space peptides by A. P. Il’ina; O. G. Kulikova; D. I. Maltsev; M. S. Krasnov; E. Yu. Rybakova; V. S. Skripnikova; E. S. Kuznetsova; A. K. Buryak; V. P. Yamskova; I. A. Yamskov (pp. 118-122).
We performed the matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF) analysis of the peptides entering into the composition of not yet explored bioregulators derived from the extracellular matrix of the tissues of the various organs of the mammals, and also plants and fungi. The study included 15 different mammalian tissues, 13 species of plants, and 2 species of fungi. Exploring the bioregulators derived from eye tissues, we demonstrated that their composition includes peptide components with the same values of the molecular weight. The composition of the bioregulators derived from the tissues of various organs of mammals or different species of plants and fungi includes the peptides with different values of molecular weight. Obtained data indicate the growing evidence of the assumptions about the major function of the bioregulators of this group—their involvement in the regulation of tissue-organ homeostasis in the biological systems.
Cloning, expression, and isolation from Escherichia coli of human protein SURF-6 translationally fused to glutathione-S-transferase by M. Yu. Kordyukova; O. V. Zatsepina; M. A. Polzikov (pp. 123-127).
cDNA of human gene Surf-6 (hSutf-6) was amplified and cloned into vector pGEX-2T for the expression in the bacterial system of protein hSURF-6 translationally fused to glutathione-S-transferase. The resulting vector is named as pGEX-2T-GST-hSurf-6. Superproducer of chimeric protein GST-hSURF-6 was obtained on the basis of Escherichia coli strain BL21-CodonPlus(DE3)-RIL. Its purification was performed by the affinity chromatography on L-glatathione-sepharose. The proportion of recombinant protein GST-hSURF-6 in the optimized conditions was not less than 15% of the total bacterial protein, and up to 7 mg of the protein was isolated from 1 liter of culture of the producer strain. The final fraction of eluate contained approximately 80% of GST-hSURF-6. The amount and the purity of the isolated protein were sufficient to immunize animals and obtain antibodies. Protein GST-hSURF-6 can also be used as an affinity ligand for revealing protein partners of hSURF-6 in human cells.
Study of a new group of bioregulators isolated from the greater plantain (Plantago major L.) by M. S. Krasnov; V. P. Yamskova; D. V. Margasyuk; O. G. Kulikova; A. P. Il’ina; E. Yu. Rybakova; I. A. Yamskov (pp. 128-135).
Proteins with physicochemical properties and biological activity similar to those of membranotropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.
An activated by cobalt alkaline aminopeptidase from Bacillus mycoides by U. Jankiewicz; A. Wnuk (pp. 136-143).
An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co2+ ions activated the enzyme, while Zn2+, Cu2+, Hg2+ and Mn2+ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.
Recombinant glycerol dehydratase from Klebsiella pneumonia XJPD-Li: induction optimization, purification and characterization by X. L. Xu; G. L. Zhang; B. Lv; Y. -J. Yuan; C. Li (pp. 144-150).
Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3-propanediol from glycerol. The optimization of inducting process for recombinant GDHt from Klebsiella pneumoniae XJPD-Li carried out to increase specific activity and ratio of soluble form. The optimum condition was inducing under the isopropyl-β-D-thiogalactoside concentration of 0.8 mM and the temperature of 20°C for 3 h. Homogeneity of GDHt then was obtained by affinity chromatography, resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of GDHt were pH 8.0 and 45°C, respectively. The K m for glycerol, 1,2-propanediol, 1,2-ethanediol and coenzyme B12 were 0.48 mM, 1.43 mM, 3.07 mM, and 10.03 nM, respectively. The GDHt showed relatively stable even under temperature of 40°C and a bit blunt to oxygen. The thermo-inactivation kinetic models were fit linear under different temperatures.
Immobilization of a recombinant strain producing glucose isomerase inside SiO2-xerogel and properties of prepared biocatalysts by G. A. Kovalenko; L. V. Perminova; T. V. Chuenko; L. I. Sapunova; E. A. Shlyakhotko; A. G. Lobanok (pp. 151-157).
An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.
Adsorptive immobilization of rhodococcal cells in hydrophobized derivatives of wide-pore poly(acrylamide) cryogel by M. S. Kuyukina; I. B. Ivshina; E. V. Rubtsova; R. V. Ivanov; V. I. Lozinsky (pp. 158-164).
Adsorption of Rhodococcus ruber cells on columns with poly(acrylamide) cryogel (cryoPAAG) partially hydrophobized by different quantities (0.2, 1, and 5, mol %) of chemically grafted n-dodecane residues has been studied. The adsorption capacity (1.1 × 109 cells/g) of gel carrier for rhodococcal cells and the optimal content (1 mol %) of hydrophobizing groups were determined. The respirometric method showed the high catalytic activity and functional stability of immobilized bacterial cells. Respiratory activity of immobilized rhodococci in the presence of a model mixture of oil hydrocarbons exceeded the respective parameter for free cells by 12–17%. Viability of rhodococcal cells adsorptionally fixed in hydrophobized cryoPAAG was maintained at a level of 93–95% after a half-year period of storage. The results may be used for development of immobilized biocatalyst for directed transformation of hydrocarbon compounds and biological purification of oil-polluted water.
Differential medium for revelation of bacterial producer strains of L-asparaginases by M. V. Pokrovskaya; V. S. Pokrovskii; N. N. Sokolov (pp. 165-168).
A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6–7 ml of glycerol, 4 g of L-asparagine, 0.2 g of CaCO3, and diagnostic components: 3 ml of 0.2 M CuSO4 · 5H2O and 2.5 ml of 0.1 M K3Fe(CN)6, pH 7.6–7.8. The results were counted 12–20 or 24–48 h after strain growth at 37°C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1–3.0 MU/mg of protein.
Anaerobic growth ability and alcohol fermentation activity of microscopic fungi by A. V. Kurakov; K. S. Khidirov; V. S. Sadykova; D. G. Zvyagintsev (pp. 169-175).
The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid (1.2–4.7%) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp.
Ion-exchange properties of cell walls of red seaweed Phyllophora crispa by N. R. Meichik; N. I. Popova; Yu. I. Nikolaeva; I. P. Yermakov; A. N. Kamnev (pp. 176-181).
Research into ion-exchange properties of cell walls isolated from thallus of red seaweed Phyllophora crispa was carried out. Ion-exchange capacity and the swelling coefficient of the red alga cell walls were estimated at various pH values (from 2 to 12) and at constant ionic strength of a solution (10 mM). It was established that behavior of cell walls as ion-exchangers is caused by the presence in their matrix of two types of cation-exchange groups and amino groups. The amount of the functional group of each type was estimated, and the corresponding values of pK a were calculated. It can be assumed that ionogenic groups with pK a ∼5 are carboxyl groups of uronic acids, and ionogenic groups with pK a ∼7.5 are carboxyl groups of the proteins. Intervals of pH in which cation-exchange groups are ionized and can take part in exchange reactions with cations in the environment are defined. It was found that protein was a major component of cell wall polymeric matrix because its content was 36%.
The effect of physiologically active compounds on the production of ethylene and the activity of polygalacturonase inhibiting protein in fruits by E. A. Bulantseva; M. A. Protsenko; A. S. Toropkina; N. P. Korableva (pp. 182-188).
The treatment of apple and banana fruits with 2-CEFA and ethacyde induced the production of ethylene and accelerated the ripening and accumulation of ACC in apple fruits. Inhibitors AOA, AVG, and CoCl2 acted at the different steps of ethylene biosynthesis, inhibited the physiological aging process and increased storage longevity. Treatment with astaxantine and BOA delayed the pick of ethylene production by fruits. The content of PGIP was correlated with intensity of ethylene production. The infection of fruits with phytopathogenic microorganisms lowered as the result of the inhibition of pathogen PG. The dynamics of PGIP activity in fruits suggests its important role in the processes of ripening.
Medicinal plants: Concentrators and superconcentrators of copper and its role in metabolism of these species by M. Ya. Lovkova; G. N. Buzuk (pp. 189-195).
With the combination of the atomic absorption method and spectrophotometry, we conducted the testing of medicinal plants of Russian flora (approximately 200 species) on the content of copper (Cu). We revealed 36 species—concentrators and superconcentrators of this element. The capability of these species to accumulate Cu is compared with the synthesis of physiologically active compounds (PAC), among which alkaloids and phenolic compounds prevail. The stimulating influence of Cu on the formation and accumulation of alkaloids of main structural types—derivatives of chinolysidine, isochinoline, tropane, and indole—is established. The data about the role of Cu-containing enzymes in the metabolism of alkaloids, as well as of phenolic compounds, are reviewed on the example of flavonoids. The role of concentrated copper in the medicinal effect of medicinal plants and, thus, the appearing perspective to widen their application spectrum, especially in the cases when the orientation of the action of PAC and Cu are different, is discussed.
Glucose oxidase-polypyrrole electrodes synthesized in p-toluenesulfonic acid and sodium p-toluenesulfonate by G. Ozyilmaz; A. T. Ozyilmaz; F. Can (pp. 196-205).
Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0–1.0 mM glucose substrate for both electrodes, I max value of Pt/PPy-GODNapTS electrode was approximately twice as high as that of Pt/PPy-GODpTSA electrode as 25.4 and 14.2 μA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectropho-tometrically using glucose kit. Results revealed that Pt/PPy-GODNapTS electrode exhibited better biosensor response.
The use of real-time PCR technology to assess the effectiveness of methods of DNA extraction from cultures of acidophilic chemolithotrophic microorganisms by S. V. Rogatykh; A. A. Dokshukina; T. S. Khainasova; S. V. Muradov; I. A. Kofiadi (pp. 206-210).
Comparative evaluation of efficiency of several methods of DNA extraction from storage cultures of acidophilic chemolithotrophic microorganism communities isolated from sulfide ores of Shanuch ore deposit (Kamchatka peninsula) was conducted. DNA extraction methods in various combinations of physical (heating to 65–98°C, grinding with SiO2 particles), enzymatic (treatment with lysozyme and proteinase K), and chemical (GuSCN, CTAB and KOH) treatments were tested. The evaluation of efficiency was performed using Real-time PCR. The best result was obtained for the combined method based on GuSCN lysis activity (lysis at 65°C) followed by purification with phenol and chloroform.
Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system by D. D. Mamaev; D. A. Khodakov; E. I. Dementieva; I. V. Filatov; D. A. Yurasov; A. I. Cherepanov; V. A. Vasiliskov; O. V. Smoldovskaya; D. V. Zimenkov; D. A. Gryadunov; V. M. Mikhailovich; A. S. Zasedatelev (pp. 211-220).
A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol mixtures for dissolving freeze-dried buffer components, washing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, executes heating, mixing of reagents, and movement of solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.
Keywords: nucleic acids; disposable microfluidic module; device for automated extraction and purification of nucleic acids; biochips
Microchip analytic system for multiplex analysis by real-time polymerase chain reaction with reagents immobilized in microreactors by D. V. Navolotskii; A. V. Perchik; I. A. Mark’yanov; A. A. Ganeev; M. N. Slyadnev (pp. 221-227).
A microchip analytic system that uses a silicon chip with immobilized in microreactor test-system for multiplex analysis of DNA by real-time polymerase chain reaction (real time PCR) was developed and optimized. We suggested the method of immobilization of PCR-components of a test-system, chose the stabilizer, and conducted the optimization of the composition of reaction mixture to achieve permanent stability of a microchip. We conducted optimization of preparation of samples using magnetic sorbent and indicated that, with 2.6 × 104 copies/ml, 60 min are necessary to obtain positive identification including time for preparation of model samples. The abilities of the created system were demonstrated on the example of microchip analysis of samples with different content of DNA, low absolute limits of detection (20 DNA copies in microreactor), and high reproducibility of the analysis.