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Applied Biochemistry and Microbiology (v.45, #2)
The microbial synthesis of flavin nucleotides: A review by V. Yu. Yatsyshyn; D. V. Fedorovych; A. A. Sibirny (pp. 115-124).
Recent data on the synthesis and hydrolysis of flavin nucleotides in yeast and bacteria and the regulation of this process are summarized. Specific examples are provided and the prospects of the use of genetically modified microorganisms for the industrial manufacturing of flavin mononucleotide and flavin adenine dinucleotide are considered.
Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan by I. A. Veselova; A. V. Kireiko; T. N. Shekhovtsova (pp. 125-129).
The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.
Transglycosylation of benzo[h]quinazolines by A. A. Markosyan; L. A. Abelyan; A. I. Markosyan; V. A. Abelyan (pp. 130-136).
This was the first study to apply cyclodextrin glucanotransferases (CGTase, EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli as well as pullulanase, β-amylase, β-galactosidase, and β-fructofuranosidase for transglycosylation of benzo[h]quinazolines. The combination of CGTase produced by Bacillus stearothermophilus ST-88 and γ-cyclodextrin (CD) used as a donor of glucosyl residues was the most efficient. The derivatives obtained are water-soluble. The synthesized products have been purified by various chromatographic methodsa and their fine structures have been determined.
The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica by O. A. Leonovich; Yu. A. Kurales; T. A. Dutova; E. P. Isakova; Y. I. Deryabina; Ya. M. Rabinovich (pp. 137-142).
Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.
Cloning of the Penicillium canescens endo-1,4-β-glucanase gene egl3 and the characterization of the recombinant enzyme by A. M. Chulkin; D. S. Loginov; E. A. Vavilova; A. R. Abyanova; I. N. Zorov; S. A. Kurzeev; O. V. Koroleva; S. V. Benevolensky (pp. 143-149).
The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K m and V m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.
Properties of two endoglucanases from a mutant strain Trichoderma sp. M7 with potential application in the paper industry by S. D. Petrova; N. G. Bakalova; D. N. Kolev (pp. 150-155).
Two endoglucanases were purified to electrophoretic homogeneity from the culture filtrate of a mutant strain Trichoderma sp. M7. EG-III and EG-IV had Mr of 49.7 and 47.5 kDa, and estimated pi values of 3.7 and 6.35, respectively. The optimal pH and temperature values were determined to be pH 5.0 and 60°C for the first cellulase, whereas pH 5.2 and 50 °C were optimal for the other. Endoglucanases exhibited typical Michaelis-Menten kinetics with K m and V values of 2.9 mg ml−1 and 60498.5 μmol min−1 mg−1 for EG-III and 3.8 mg ml−1 and 22650.9 μmol min−1 mg−1 for EG-IV, respectively. Mn2+, Cu2+ and Pd2+ strongly inhibited the enzymes. EC-IV catalyzed the hydrolysis of Na-CMC and hydroxyethyl cellulose (HEC) only, whereas EG-III displayed high activity towards xylans, also. Different preferences towards cellulosic substrates and their regions define a different role of the investigated enzymes in the degradation of plant biomass.
High-level secretory production of recombinant human platelet-derived growth factor-BB by Saccharomyces cerevisiae under the non-selective conditions by Y. Wang; L. Xue; Y. Li; Y. Zhu; B. Yang; X. Wang (pp. 156-161).
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) was produced by Saccharomyces cerevisiae. A two-stage cultivation strategy with mixture of glucose and galactose was developed to enhance rhPDGF-BB production, with its concentration being 32 mg/l fermentation broth under optimal conditions: corn steep powder as nitrogen source, 2 g/1 of glucose concentration at the beginning of induction phase, a pH of 5.0 and 6.5 for cell growth and rhPDGF-BB accumulation. The purification process consisted of yeast supernatant ultrafiltration followed by ion exchange chromatography and molecular sieve, and the recovery of rhPDGF-BB was estimated to be 20.28%. Biological activity of the purified rhPDGF-BB was 3.05 ng/ml.
Effect of, hexylresorcinol, a chemical analogue of bacterial anabiosis autoinducers on the stability of membrane structures by N. G. Loiko; A. L. Mulyukin; A. N. Kozlova; A. P. Kaplun; V. V. Sorokin; I. A. Borzenkov; Yu. A. Nikolaev; A. S. Kaprel’yants; G. I. El’-Registan (pp. 162-168).
The effect of hexylresorcinol (HR), a chemical analogue of microbial anabiosis autoinducers of the alkylhydroxybenzene (AHB) group, on the stability of biological membranes and monolamellar liposomes formed of egg phosphatidylcholine (ePC) was studied. According to spectrophotometry and electron microscopy studies of HR-loaded liposomes in the presence of a surfactant Tween 20, the critical ratio between HR and ePC for liposome preservation was found to be close to equimolar. The trends in HR influence on membrane structural organization and stability of liposomes were also confirmed in experiments on intact bacterial cells explaining non-species-specific effect of AHBs. The demonstrated high efficiency of AHB biocides may be used in material and equipment protection against biocorrosion.
Phenanthrene and anthracene degradation by microorganisms of the genus Rhodococcus by N. A. Leneva; M. P. Kolomytseva; B. P. Baskunov; L. A. Golovleva (pp. 169-175).
The cells of Rhodococcus opacus 412 and R. rhodnii 135 were adapted to phenanthrene and anthracene on a solid mineral medium. Preliminary adaptation of the strains accelerated the metabolism of polyaromatic hydrocarbons and provided for the ability of microorganisms to grow on pheanthrene as a sole carbon and energy source in a liquid mineral medium. It was shown that phenanthrene was mineralized by the strains through 7,8-benzocoumarin, 1-hydroxy-2-naphthoaldehyde, 1-hydroxy-2-naphthoic acid, salicylaldehyde, salicylate and catechol to the intermediates of tricarbonic acid cycle and partially transformed with the accumulation of the products of subsequent monooxygenation (3-hydroxyphenanthrene and phenanthrene dihydroxylated not in ortho-position). As a result of the adaptation of the strains to anthracene on a solid mineral medium, the obtained variant of strain R. opacus 412 was able to transform anthracene in a liquid mineral medium to anthraquinone and 6,7-benzocoumarin.
Special traits of decomposition of azo dyes by anaerobic microbial communities by N. A. Yemashova; I. B. Kotova; A. I. Netrusov; S. V. Kalyuzhnyi (pp. 176-181).
We present the results of an investigation into the special traits of conversion of azo dyes Acid Orange 6, Acid Orange 7, Methyl Orange, and Methyl Red under anaerobic conditions in comparison to aerobic conditions. In the presence of oxygen, only Methyl Red underwent decomposition, while under oxygen-free conditions, all remaining substances were fully decolourised under the action of a methanogenous consortium of microorganisms. The products of reduction of the azo bond are determined in the case of each dye. Introduction of additional acceptors of electrons (sulfate and nitrate) had a negative influence on the discoloration of azo dyes. Addition of ethanol as an available organic cosubstrate accelerated decomposition of azo dyes both under methanogenous and sulfate- and nitrate-reducing conditions. There is no direct correlation between the rates of conversion of azo dyes under anaerobic conditions or their toxicity to acetoclastic methanogens. Changes in the morphological composition of the community decolouring an azo dye depended on the duration of its impact on microorganisms. The mechanism of the reduction of the azo bond under the action of substances acting as mediators is explained. These substances are products of the metabolism of the microbial community in anaerobic conditions. It is shown that the supposed mediators NADH and sulfide efficiently decolourise azo dyes in a cell-free system, while riboflavin significantly increased the rate of conversion of substrates in recurrent cycles of discoloration only in the presence of an anaerobic microbial consortium.
Clavine alkaloid biosynthesis by the fungus Penicillium palitans Westling 1911 isolated from ancient permafrost deposits by A. G. Kozlovsky; V. P. Zhelifonova; T. V. Antipova (pp. 182-186).
The relic strain of Penicillium palitans isolated from the ancient permafrost deposits produces clavine alkaloids such as festuclavine, fumigaclavine A, and fumigaclavine B. Alkaloid biosynthesis is concurrent with the growth. Tryptophan and zinc ion additives to the culture medium stimulate the synthesis of alkaloids.
The composition of volatile components of cepe (Boletus edulis) and oyster mushrooms (Pleurotus ostreatus) by T. A. Misharina; S. M. Muhutdinova; G. G. Zharikova; M. B. Terenina; N. I. Krikunova (pp. 187-193).
The composition of aroma compounds in cooked and canned cepe (Boletus edulis) and in cooked oyster mushrooms (Pleurotus ostreatus) is studied using capillary gas chromatography and chromatographymass spectrometry. It is found that unsaturated alcohols and ketones containing eight atoms of carbon determine the aroma of raw mushrooms and take part in the formation of the aroma of cooked mushrooms as well. The content of these compounds was the highest in canned cepes. In oyster mushrooms, the concentration of these alcohols and ketones was lower in comparison with cepes. The content of aliphatic and aromatic aldehydes was much higher in oyster mushrooms. Volatile aliphatic and heterocyclic Maillard reaction products and isomeric octenols and octenones formed the aroma of cooked and canned mushrooms.
Evaluation of the effect of late blight-resistant potato plants on the structure of bacterial associations in soil by E. V. Zadorina; E. S. Boulygina; T. V. Kolganova; B. B. Kuznetsov; K. G. Skryabin (pp. 194-198).
We studied the compositions of microbial associations isolated from soils where nontransgenic and transgenic late blight-resistant lines of potato varieties Lugovskoi, Charodei, and Golubizna had been grown. The analysis was based on denaturing gradient gel electrophoresis of total amplificates of 16S rRNA gene fragments and analysis of clone libraries of nifH gene fragments. Neither method revealed significant differences in the structure of the microbial associations isolated from soils with control or transgenic plants. The minor differences detected in the microflora ranges were no greater than those in the rhizospheres of different nontransgenic potato varieties.
Wound healing and induced resistance in potato tubers by O. L. Ozeretskovskaya; N. I. Vasyukova; G. I. Chalenko; N. G. Gerasimova; T. A. Revina; T. A. Valueva (pp. 199-203).
It was demonstrated that biogenic elicitors, arachidonic acid and chitosan, locally and systemically stimulated wound healing in potato tuber tissues by increasing the number of wound periderm layers, accelerating the development of cork cambium (phellogen), and inducing proteinase inhibitors. The signal molecules, jasmonic and salicylic acids, had different effects on the development of wound periderm: jasmonic acid locally and systemically stimulated potato wound healing and elevated the level of proteinase inhibitors, whereas salicylic acid did not have any effect on wound healing and even blocked the formation of proteinase inhibitors.
Development of immunochromatographic test systems for express detection of plant viruses by N. A. Byzova; I. V. Safenkova; S. N. Chirkov; A. V. Zherdev; A. N. Blintsov; B. B. Dzantiev; I. G. Atabekov (pp. 204-209).
Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rodshaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 μg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.
Enzyme immunoassay for the determination of the glycopeptide antibiotic eremomycin by M. A. Burkin; A. A. Burkin (pp. 210-214).
An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using rabbit polyclonal antibodies against the eremomycin-glucose oxidase conjugated antigen. This technique allows the glycopeptide antibiotic eremomycin to be determined both in aqueous solutions (with a sensitivity as high as 0.1 ng/ml) and in blood plasma. The cross-reactivity of the antibodies with vancomycin was 0.4% of that for eremomycin, while teicoplanin was almost not recognized. Experiments with blood plasma samples diluted 1: 10 showed that the assay was linear over the concentration range 1–30 ng/ml and that the variation coefficient did not exceed 10%. The high sensitivity and selectivity of this test make it suitable for pharmacokinetic studies and drug monitoring analysis.
Affine magnetic sorbents supported on coal ash microspheres for recombinant protein isolation by L. A. Frank; V. V. Borisova; T. A. Vereshchagina; E. V. Fomenko; A. G. Anshits; I. I. Gitelson (pp. 215-220).
The results of the development and utilization of an affine magnetic sorbent with Ni2+ ions immobilized on coal ash microspheres are reported. The applicability of the material in the isolation of Histag proteins is demonstrated by examples of the recombinant green fluorescent protein from Clytia gregaria and the Ca2+ regulated photoprotein obelin from Obelia longissima. The specific sorption capacity of the sorbent was 2–7 mg/cm3 for medium-size proteins (20–30 kDa). The particles are suitable for chromatography with the presence of chaotropic agents and EDTA. They are easy to manipulate as isolation of a target protein takes 30–35 min. On the one hand, the elevated affinity of the sorbent to proteins rich in native histidines may result in a high degree of irreversible sorption; on the other hand, it allows isolation of such proteins without the introduction of artificial polyhistidine fragments.
Sorption of lysine by molecularly imprinted carboxyl sorbents by N. M. Ezhova; I. V. Polyakova; O. A. Pisarev (pp. 221-225).
Tuned (molecularly imprinted) and nontuned, with respect to lysine amino acid, carboxylic heteroreticular sorbents based on methacrylic acid and ethylene glycol dimethacrylate, were synthesized. Study of sorption of lysine within wide pH range and ionic strength indicated significant dissimilarities in amino acid sorption by tuned sorbents, which were expressed as an increase in the contribution of nonionic interaction, and resulted in a decrease in the ionic strength effect on the sorption capacity, as well as an increase in amino acid sorption selectivity.
Antimicrobial activity of carbon fiber fabric modified with a polymer-gentamicin complex by M. V. Solovskii; V. I. Dubkova; N. P. Krut’ko; E. F. Panarin; M. Yu. Smirnova; N. A. Belyasova; O. I. Maevskaya (pp. 226-228).
Modified and unmodified carbon fiber supports were treated with solutions of a polymer-gentamicin complex, possessing high antimicrobial activity and low toxicity. It was found that the antimicrobial activity of modified carbon fiber fabrics depended on the nature of the support and on the immobilization conditions. The highest antimicrobial activity was observed with phosphorus-containing carbon fiber ion exchanger in salt form.
Carbon nanotube-based biosensors for DNA structure characterization by T. I. Abdullin; O. V. Bondar’; A. A. Rizvanov; I. I. Nikitina (pp. 229-232).
The possibility of DNA detection using electrodes modified with carbon nanotubes (CNTs) was studied. CNTs facilitate the electrochemical oxidation of DNA guanine nucleotide, which allows direct detection of DNA on a modified electrode. Electrochemical properties of DNA depend on its secondary structure and molecular weight. Denaturation of native DNA improves the adsorption of biopolymer on CNTs and results in an increase in DNA oxidation current on the modified electrode. A similar effect is observed after ultrasonic shearing of DNA or its treatment with Fenton’s reagent due to the fragmentation of biopolymer. Our results demonstrate the feasibility of biosensors based on CNT-modified electrodes for the direct detection and characterization of DNA and DNA damaging factors.