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Applied Biochemistry and Microbiology (v.46, #2)

Rediscovering cyanobacteria as valuable sources of bioactive compounds (Review) by R. Prasanna; A. Sood; P. Jaiswal; S. Nayak; V. Gupta; V. Chaudhary; M. Joshi; C. Natarajan (pp. 119-134).

Cyanobacteria are a simple, but primitive and diverse group of microorganisms, with characteristics in common to both bacteria and algae. Their success as a group in a wide range of habitats has been attributed to their unique physiological characters and high adaptive ability under a wide range of environmental conditions. The potential of cyanobacteria as a source of a variety of compounds such as polysaccharides, lipids, proteins, vitamins, sterols, enzymes, pharmaceuticals and other fine chemicals is well recognized, and their demand is now on an increasing trend. This compilation reviews the salient advances in the discovery of bioactive compounds from cyanobacteria and their significance in agriculture and industry.

Lecithin influence on the effectiveness of the antioxidant effect of flavonoids and α-tocopherol by L. I. Mazaletskaya; N. I. Sheludchenko; L. N. Shishkina (pp. 135-139).

Influence of the widely used food additive lecithin on the effectiveness of the inhibiting effect of the natural antioxidants (quercetin, dihydroquercetin, and α-tocopherol) has been studied in dependence on the rate of free radicals generation in the model oxidation reactions. It has been found that during the initiated and autoxidation of methyl oleat, lecithin decreased the antioxidant effectiveness of flavonoids. The effect value increased with the lecithin concentration increase. Under similar conditions while oxidation inhibiting by α-tocopherol and lecithin mixtures, the latter did not influence the tocopherol antioxidant effectiveness (additivity) or led to the increase of the inhibition effectiveness (synergism).

Cloning and molecular organization of the polyhydroxyalkanoic acid synthase gene (phaC) of Ralstonia eutropha strain B5786 by I. V. Kozhevnikov; T. G. Volova; Tran Hai; A. Steinbüchel (pp. 140-147).

Class I polyhydroxyalkanoic acid (PHA) synthase gene (phaC) of Ralstonia eutropha strain B5786 was cloned and characterized. R. eutropha B5786 features the ability to synthesize multicomponent PHAs with short- and medium-chain-length monomers from simple carbohydrate substrate. A correlation was made between the molecular structure of PHA synthase and substrate specificity and the ability of strain-producers to accumulate PHAs of this or that structure. A strong similarity of PHA synthase of R. eutropha strain B5786 with PHA synthase of R. eutropha strain H16, which, as opposed to strain B5786, enables to incorporate medium chain length PHAs if hexanoate is used as carbon source, exhibited 99%. A correlation between the structure of PHA synthase of B5786 strain with synthases of microorganisms which synthesize short and medium chain length PHAs similarly to B5786 strain, showed an identity level from 26 to 41% (homology with synthase of Rhodospirillum rubrum makes 41%, Ectothiorhodospira shaposhnikovii makes 26%, Aeromonas punctata makes 40%, Thiococcus pfennigii makes 28%, Rhodococcus ruber makes 38%, and with PhaCl and PhaC2 synthases of Pseudomonas sp. 61–3 makes 34 and 37%, respectively). This allows for speaking about the absence of a direct connection between the molecular organization of PHA synthases and their functional abilities, namely, the ability to synthesize PHAs of a particular composition.

Function analysis of a new type I PKS-SAT domain by Sat-Eat domain replacement by Y. L. Jiao; L. H. Wang; B. H. Jiao; S. J. Wang; Y. W. Fang; S. Liu (pp. 148-153).

The function of a new starter unit acyltransferase (SAT) domain SAT-EF080951 (GenBank accession number) encoded in a new type I polyketide synthase (PKS) gene cluster EF568935 (GenBank accession number) isolated for this study was analyzed by domain replacement with an extender unit AT (EAT) domain of avermectin PKS. It was shown that the SAT-EF080951 incorporated malonyl-CoA specifically in vivo, which contradicted the specificity that we had previously determined by substrate binding test in vitro. The result of this study indicates that type I PKS-SAT can alter its specificity in vivo and functions well in extender units and proved the feasibility of the SAT-EAT domain replacement in type I PKS. We propose that SAT-EAT replacement strategy could be a novel route for increasing the diversity of new polyketides combinatorially biosynthesized. The new type I PKS-SAT-EF080951 studied herein may be further employed for related studies on enzymology or combinatorial biosynthesis of polyketides.

Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein by L. R. Arslanbaeva; V. V. Zherdeva; T. V. Ivashina; L. M. Vinokurov; A. L. Rusanov; A. P. Savitsky (pp. 154-158).

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within the FRET-pair, the engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21(DE3) was studied and conditions of synthesis, isolation, and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic diffusion. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of fluorescence spectroscopy. The obtained FRET-pair may be used both for studies in vitro and as reporters in living cells.

Antimicrobic features of phenolic lipids by Yu. A. Nikolaev; I. A. Borzenkov; M. V. Kalinin; N. G. Loiko; A. L. Tarasov; V. K. Plakunov; S. S. Belyaev; N. V. Voronina; V. F. Gal’chenko; G. I. El’-Registan (pp. 159-165).

The connection between the efficiency of phenolic lipids and their hydrophobic property (solubility) and hydrophobic property of microorganisms’ cell structure is shown. The mixture of amphiphilic di(oxiphenil)-phenil-methanes, which act bacteriostatically under 15 mg/l, possesses maximal efficiency against Staphylococcus aureus. Against Mycobacterium smegmatis with hydrophobic cell wall, hydrophobic 2,4-dialkylocibenzol 70 mg/l was the most effective. Hexylresorcin (HR) stops the development of gram-positive bacteria in concentrations 20–50 mg/l, that of gram-negative bacteria in concentration 65 mg/l, that of M. smegmatis at 70 mg/l, and that of yeast and fungi at 300 mg/l. HR prevails bacteria spores germination in the concentration 25–100 mg/l. The dependence of antibacterial action of isomers and homologues of alkylresorcins on their structure-number, position, and length of alkyl substituents-is studied.

Stearic acid methyl ester: A new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei by E. A. Terekhova; N. A. Stepicheva; A. B. Pshenichnikova; V. I. Shvets (pp. 166-172).

Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C₁₆–C₁₈ and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C_{18: 1} > C_{18: 0} > C_{16: 0} > methyl oleate > methyl stearate > no supplements > C_{14: 0} > C_{12: 0}. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.

Biosynthesis of the bioprotectant ectoin by aerobic methylotrophic bacteria from methanol by N. V. Doronina; V. A. Ezhov; A. P. Beschastnyi; Yu. A. Trotsenko (pp. 173-176).

It is shown that neutrophilic methylobacteria Methylophaga thalassica and M. marina have higher rates of growth and ectoin accumulation compared to the haloalkaliphilic species M. alcalica and M. natronica and methanotrophs Methylomicrobium alcaliphilum and M. kenyense. The conditions of M. thalassica cultivation in methanol-containing medium were optimized. The yield of this process reached 60 g/l of absolutely dry biomass containing 15–19% (9–11 g/l) ectoine. The scheme of ectoin isolation from the biomass by extraction and subsequent purification, which allowed obtaining preparations of different degree of purity, was developed.

Protective and reactivating effect of the protein exometabolite on yeast cells inactivated by the ultraviolet irradiation by L. I. Vorob’eva; E. Yu. Khodzhaev (pp. 177-183).

It has been shown that Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida utilis strains produce the protein exometabolites, which has a protective and reactivating effect on the ultraviolet irradiated yeast cells. The protective effect of the preliminary ultraviolet irradiated (activated) protein exometabolite of all strains increased 2–3 times, though its reactivating activity did not change. Yarrowia lipolytica yeast cells, isolated from the areas with the high daily irradiation, and Endomyces magnusii, the obligate fungi parasites, were characterized by the highest ultraviolet tolerance in comparison with the other strains. However, they did not produce the exometabolites with the antistress effect. Luteococcus casei reactivating factor demonstrated protective and reactivating cross-action in relation to the ultraviolet irradiated S. cerevisiae, K. lactis, and C. utilis cells and were inactive in relation to Y. lipolytica and E. magnusii. Using killer and nonkiller S. cerevisiae strain, it has been shown that the peptide exometabolite accumulation was not associated with toxin production.

The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum by A. Ya. Valiakhmetov; L. V. Trilisenko; V. M. Vagabov; Yu. E. Bartoshevich; I. S. Kulaev; M. I. Novak; A. G. Domracheva; M. A. El’darov; K. G. Skryabin (pp. 184-190).

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H⁺-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyP5 with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H⁺-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.

Characterization of hydrocortisone bioconversion and 16S RNA gene in Synechococcus nidulans cultures by S. Rasoul-Amini; Y. Ghasemi; M. H. Morowvat; M. B. Ghoshoon; M. J. Raee; S. B. Mosavi-Azam; N. Montazeri-Najafabady; F. Nouri; R. Parvizi; N. Negintaji; S. Khoubani (pp. 191-197).

A unicellular cyanobacterium, Synechococcus nidulans (Pringsheim) Komárek, was isolated from paddy-fields and applied in the biotransformation experiment of hydrocortisone (1). This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25°C for 14 days of incubation. The products obtained were chromatographically purified followed by their characterization using spectroscopic methods, 11β,17β-dihydroxyandrost-4-en-3-one (2), 11β-hydroxyandrost-4-en-3,17-dione (3), and androst-4-ene-3,17-dione (4) were the main bioproducts in the hydrocortisone bioconversion. The observed bioreaction characteristics were the side chain degradation of the substrate to prepare compounds (2) and (3) following the 11β-dehydroxylation for accumulation of the compound (4). Time course study showed the accumulation of the product (2) from the second day of the fermentation and compounds (3) and (4) from the third day. All the metabolites reached their maximum concentration in seven days. Cyanobacterial 16S rRNA gene was also amplified by PCR. Sequences were amplified using the universal prokaryotic primers which amplify a ∼400-bp region of the 16S rRNA gene. PCR products were sequenced to confirm their authenticity as 16S rRNA gene of cyanobacteria. The result of PCR blasted with other sequenced cyanobacteria in NCBI showed 99% identity to the 16S small subunit rRNA of seven Synechococcus species.

Bioconversion of C₁₉- and C₂₁-steroids with parent and mutant strains of Curvularia lunata by V. V. Kollerov; A. A. Shutov; V. V. Fokina; G. V. Sukhodol’skaya; S. A. Gulevskaya; M. V. Donova (pp. 198-205).

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (¹H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C₂₁-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.

The effect of point amino acid substitutions in an internal α-helix on thermostability of Aspergillus awamori X100 glucoamylase by M. A. Surzhik; S. V. Churkina; A. E. Shmidt; A. V. Shvetsov; T. N. Kozhina; D. L. Firsov; L. M. Firsov; M. G. Petukhov (pp. 206-211).

Conformational flexibility of α-helices in glucoamylase of the fungus Aspergillus awamori was studied by molecular dynamics methods. Several amino acid substitutions (G127A, P128A, I136L, G137A, and G139A) optimizing intrinsic interactions in one of the α-helices (D) within the hydrophobic core of this protein were constructed and studied. It was found that these point mutations had different effects on the glucoamylase thermal inactivation constant. Unlike amino acid substitution P128A and substitutions G137A and A246C, I136L and G139A displayed a pronounced additive thermostabilizing effect.

Interaction of phytohormones and synthetic growth regulator melafen in the control of Ca²⁺ translocation across the plasma membrane of potato (Solanum tuberosum L.) tuber cells by E. P. Ladyzhenskaya; N. P. Korableva (pp. 212-215).

Interference of phytohormones (jasmonic, gibberellic, and abscisic acids) and synthetic growth regulator melafen on Ca²⁺ translocation across the membrane of plasma membrane vesicles prepared from dormant potato (Solanum tuberosum L.) tubers was studied. The activity of plasma membrane Ca²⁺, Mg²⁺-ATPase was stimulated by melafen and jasmonic and gibberellic acids and suppressed by abscisic acid. These substrances did not change the passive membrane permeability for Ca²⁺. The pattern of the effect of melafen on the activity of Ca²⁺,Mg²⁺-ATPase depended on the presence of phytohormones in incubation medium. When melafen and each phytohormone were simultaneously added to incubation medium, their effects were not additive, which indicates that the effects of the tested compounds on the Ca²⁺ uptake into the plasma membrane vesicles are interdependent. Apparently, the interaction between the phytohormones and plasma membrane components modulates the response to melafen.

Enzyme immunoassay for determination of sulfamethoxypyridazine in honey by I. Yu. Tafintseva; A. V. Zherdev; S. A. Eremin; B. B. Dzantiev (pp. 216-220).

An enzyme immunoassay technique for the detection of sulfamethoxypyridazine in honey, developed using rabbit polyclonal antibodies raised against N-sulfonyl-4-aminobutyric acid, which contains a structural group characteristic of sulfonamides, is proposed. Under the optimized conditions, the sulfamethoxypyridazine detection limit was 0.05 ng/ml, with the entire analysis procedure taking 2 h. In total, 24 honey samples were tested using the protocol based on tenfold dilutions of samples without their preliminary treatment.

Association-dissociation of molecules of hemoglobin and polymeric hemoglobin in solutions by N. P. Kuznetsova; L. R. Gudkin; R. N. Mishaeva; E. A. Berezetskaya; M. E. Vylegzhanina; T. E. Sukhanova; E. F. Panarin (pp. 221-225).

The process of association-dissociation of hemoglobin molecules into dimers of its subunits in water and water-saline solutions is studied by the method of gel-penetrating chromatography and ultrafiltration. The quantitative assessment of stabilization of quaternary structure of hemoglobin in chemically bound polymer derivative in comparison with native peptide on the basis of building differential concentration curves is conducted for the first time. By the method of atomic-force spectroscopy, the morphology of nanoparticles of hemoglobin and its modified polymeric derivative is studied.

Purification of recombinant proteins with an example of tumor necrosis factor thymosin-α1 by T. V. Fedorov; V. I. Korobov; V. G. Nazarov; A. E. Smolkina; V. A. Shmelev (pp. 226-229).

Hybrid protein, cancer necrosis factor thymosin-α1 (TNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of “inclusion bodies” mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of TNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-Na) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous TNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of TNF-T achieved 80% relative its content in biomass and 30% relative the total protein.

A new thermostable DNA polymerase mixture for efficient amplification of long DNA fragments by K. G. Davalieva; G. D. Efremov (pp. 230-234).

The thermostable DNA polymerases have been used for amplification of DNA fragments since the invention of PCR. The constraint on the maximum size of the amplified fragments can be solved to certain level by the use of unbalanced mixtures of non-proofreading and proofreading thermostable DNA polymerases. In this study, we tested the use of a mixtures of N-terminal deletional variant of Taq polymerase—Klentaq278 and Tne polymerase from Thermotoga neapolitana. Klentaq278 and Tne polymerase genes were cloned and expressed in different expression vectors under tac promoter. The most efficient ratio of Klentaq278/Tne polymerase for amplification was 10: 1. The polymerase mixture of Klentaq278 and Tne polymerase is very effective in amplification of DNA fragments for up to 8 kb and is useful addition to a DNA polymerases used in long-range PCR.

A Review of the Book Label-Free Biosensors: Techniques and Applications. Edited by Matthew A. Cooper. Cambridge, 2009 by A. N. Reshetilov (pp. 235-238).

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Applied Biochemistry and Microbiology (v.46, #2)

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Applied Biochemistry and Microbiology (v.46, #2)