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Applied Biochemistry and Microbiology (v.46, #8)

Microbial desulfurization of motor fuel by V. G. Debabov (pp. 733-738).

Deep desulfurization of oil and its fractions is currently performed by hydration at high temperature and hydrogen pressure, which makes the process rather expensive. Searches for alternative modes for desulfurization, among which is biodesulfurization, are intensely in progress. In this review, the following subjects are discussed: microorganisms capable of desulfurizing petroleum products, mechanisms of their activity, achievements in the field of process development, and disadvantages of the method. The existing level of knowledge is insufficient for immediate implementation of an industrial biotechnological process for sulfur elimination from oil and motor fuel and it can only be regarded as a medium-term (10–15 years) prospect.

Keywords: biodesulfurization; enzymes cloning; biodesulfurization technology; difficult-to-desulfurize compounds (benzothiophene and dibenzothiophene); enzymes and microorganisms involved in desulfurization

Biocompatible materials from regenerated silk for tissue engineering and medicinal therapy by A. S. Kon’kov; O. L. Pustovalova; I. I. Agapov (pp. 739-744).

The present review is devoted to the application of biomaterials from regenerated silk for designing tissue-engineered constructs—the basis for hybrid organs and tissues. Fibroin, the main structural protein of silkworm silk, can be used to design artificial cartilages, bone tissue fragments, blood vessels, as well as to regenerate nervous tissue. Fibroin capsules containing bioactive compounds are successfully applicable in medicinal therapy, such as controlled drug delivery in cancer treatment. Apart from fibroin, tissue engineering can successfully be based on biopolymer spidroin, a spider net protein, which is also a biocompatible material with valuable mechanical properties.

Keywords: artificial matrix; medicinal therapy; spider net; regenerated silk; creation of hybrid tissues and organs; transplantology; fibroin

Construction of a butyrate-producing E. coli strain without the use of heterologous genes by T. A. Seregina; R. S. Shakulov; V. G. Debabov; A. S. Mironov (pp. 745-754).

Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter P tet, and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, P tet-atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l.

Keywords: genetic construction; fatty acid β-oxidation; butyrate synthesis; E. coli

Larvicidal activity of crystal-forming strains of Brevibacillus laterosporus by M. V. Zubasheva; L. A. Ganushkina; T. A. Smirnova; R. R. Azizbekyan (pp. 755-762).

The optimum conditions for growth, sporulation, and crystal-formation in four isolated crystal-forming strains of Bacillus laterosporus were determined. It was shown that culture broth and pellets of bacterial culture liquid possess larvicidal activity against larvae of mosquitoes A. stephensi and A. aegypti. The protein nature of crystal was shown. Crystals are monocomponent containing a protein with MM of 68 or 130 kDa. Purified protein crystals demonstrated larvicidal activity. Specific larvicidal activity of crystals of various strains essentially differed. High larvicidal activity of B. laterosporus strains allows for them to be recommended as producers of antimosquito biological preparations.

Keywords: Brevibacillus laterosporus ; entomocidal crystal; larvicidal activity; mosquito

In vitro response of transgenic aspen containing glutamine synthetase gene GSI to the sublethal dose of phosphinothricin by K. A. Shestibratov; I. V. Bulatova; P. S. Novikov (pp. 763-768).

Transgenic aspen plants containing the glutamine synthetase gene GSI from pine have been produced. Among 37 transformed lines, 34 were found to possess GSI. The RT-PCR analysis of GSI transcripts confirmed the presence of specific transcripts in 32 lines. The phenotypic effect of a glutamine synthetase activity in transgenic plants was evaluated by in vitro cultivation of plants at the presence of a sublethal dose (0.5 mg/l) of phosphinothricin, which inhibits this enzyme. It was shown that the sublethal dose of this herbicide provides a predictable inhibiting effect on the nontransformed aspen plants, including the inhibition of their rhizogenesis, whereas transgenic plants demonstrated various responses. In most transgenic lines, we observed an unexpected stimulating effect of low herbicide doses on in vitro rhizogenesis; this effect was manifested through the increased radication frequency, increased average number of roots per plant, and increased total length of roots.

Keywords: agrobacterial transfer; in vitro rhizogenesis; transformant

Method of clonal micropropagation of different willow species and hybrids by O. S. Mashkina; T. M. Tabatskaya; A. I. Gorobets; K. A. Shestibratov (pp. 769-775).

Conditions of cultivation and micropropagation of selected biotypes of five willow species (Salyx dasyclados Wimm., S. caspica Pall., S. triandra L., S. purpurea L., and S. viminalis L.) and two hybrids (×S. acuminata S. and ×S. palustris Host.) were optimized. Data on in vitro propagation of S. caspica, S. triandra, S. purpurea together with hybrids S. acuminata and S. palustris were obtained for the first time. It has been demonstrated that the outcome of cultivation and propagation of willows strongly depends on genotypic peculiarities of initial plants. The optimal terms of isolation and sterilization of single-node segments for obtaining 50–75% of aseptic viable developing cultures were estimated. The nutritive media were selected providing induction of stem development (to 67%), their rooting (to 91%), elongation (to 3–6 cm), and multiplication (propagation coefficient of 4). The designed method (adopted to different genotypes) can be applied for obtaining aseptic in vitro cultures serving as initial plant material for genetic transformation and mass propagation of plants with new agriculturally valuable characteristics which are of interest for construction of bioenergetic plantations and for needs of the paper industry.

Keywords: genotype; in vitro propagation; multiplication; rooting; explant

Expression of a deleted variant of human plasminogen in Escherichia coli by Ya. G. Gurskii; M. M. Minashkin; E. S. Feoktistova; A. V. Skamrov; N. A. Skrypina; R. Sh. Bibilashvili (pp. 776-780).

A recombinant plasmid carrying a modified gene of human plasminogen (mini-plasminogen), lacking four kringle domains and an amino terminal fragment, and containing an additional oligopeptide of six N-terminal histidine residues has been constructed. The plasmid was used for transformation of E. coli JM 109 cells to obtain a strain producing a recombinant modified human plasminogen. The target protein is superexpressed in a form of inclusion bodies and is composed of more than 50% insoluble protein. The renaturated and chromatographically purified protein exhibits amidolytic activity specific for plasminogen proenzyme in a fibrinolytic system.

Keywords: bacterial producer; modified protein; plasmin; plasminogen activation; recombinant plasmid DNA; trombolysis

Lux-biosensors for detection of SOS-response, heat shock, and oxidative stress by V. Yu. Kotova; I. V. Manukhov; G. B. Zavilgelskii (pp. 781-788).

We have constructed hybrid plasmids pColD and pIbpA in which transcription of reporter genes luxCDABE from Photohabdus luminescens occurred from induced promoters: SOS-promoter of gene cdaA (pColD-CA23) and heat shock gene ibpA (E. coli MG1655 genome), respectively. Main parameters were measured: threshold concentration of an inducer and maximal response and minimal time of response for E. coli lux-biosensors bearing hybrid plasmids pColD and pRecA (SOS-response); pIbpA and pGrpe (heat shock) and pKatG and pSoxS (oxidative stress). Mitomicyn C, cis-diaminedichloroplatinum (SOS-response), ethanol and pentachlorophenol (heat shock), and hydrogen peroxide and methyl viologen (oxidative stress) were used as luminescence inducers. It was shown that the SOS-biosensor pColD is superior in main characteristics to the earlier constructed biosensor pRecA, and the heat biosensor pIbpA is more efficient than the earlier constructed pGrpE. Lux-biosensors (pKatG) and (pSoxS) are high sensitive to hydrogen peroxide and methyl viologen, respectively.

Keywords: bioluminescence; biosensor; induced promoter; Pcda; PibpA; PgrpE; PkatG; PrecA; PsoxS

Use of integrated phytoremediation for cleaning-up of oil-sludge-contaminated soil by A. Yu. Muratova; A. D. Bondarenkova; L. V. Panchenko; O. V. Turkovskaya (pp. 789-794).

The possibility of using multicomponent systems, including plants, mineral fertilizers, and plant growth promoting microorganisms, has been studied in vegetative experiments in order to stimulate phytoremediation of oil-sludge-contaminated soil. Winter rye (Secale cereale L.) was used as the principal phytoremediating plant species, whereas alfalfa (Medicago sativa L.), nitrogen fertilizer (ammonium nitrate), and a PGPR strain (Azospirillum brasilense SR80) were applied as additional components, individually or in various combinations. The obtained data revealed the critical importance of alfalfa for phytoremediation of hydrocarbon-contaminated soil. Application of different multicomponent treatments resulted in approximately 70% reduction of pollutant concentration in soil. The developed technological approaches were successfully tested in the remediation of an ex-oil-sludge pit on the ground of a petroleum refinery.

Keywords: Medicago sativa ; nitrogen fertilizer; phytoremediation; plant growth promoting rhizobacterium; Secale cereale

Enzyme-linked immunosorbent assay of chlorampenicol in foodstuff by M. D. Fedorova; I. P. Andreeva; E. S. Vilegzhanina; A. A. Komarov; M. Yu. Rubtsova; J. V. Samsonova; A. M. Egorov (pp. 795-801).

A test-system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chloramphenicol (CAP) in foodstuff has been developed. The detection limit of the method was 0.05 μg/l. The procedures for milk samples preparation of various fat content and chicken muscles were optimized. Before the analysis milk was diluted 5-fold with a buffer. The detection limit for milk was 0.3 μg/l; recoveries varied from 74 to 118%. Two protocols for chicken muscles preparation were elaborated; extraction with buffer (the express method) and extraction with acetonitrile. The detection limits of CAP in chicken muscles were 0.5 and 0.3 μg/kg, respectively; recovery values were 71–107% and 95–115%, respectively. The results of residual amounts of CAP detection in foodstuff by ELISA and HPLC-MS were in good correlation.

Keywords: enzyme-linked immunosorbent assay; food of animal origin; chloramphenicol

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Applied Biochemistry and Microbiology (v.46, #8)