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Applied Biochemistry and Microbiology (v.47, #3)
Wound repair in plant tissues (Review) by N. I. Vasyukova; G. I. Chalenko; N. G. Gerasimova; O. L. Ozeretskovskaya (pp. 229-233).
Signaling systems responsible for repair processes in plants and manifestation of defensive effects in plant tissues were analyzed. Special attention was given to jasmonic acid, a mobile systemic repair signal, as well as to jasmonate biosynthesis and signal transport to the areas where protective responses of plants are induced. The main defense responses of potato tubers induced by wounding were considered.
Identification of catalytically active groups of pea (Pisum sativum L.) β-glucosidase by A. N. Ershova; O. N. Barkalova (pp. 234-238).
Functional groups of cytoplasmic pea β-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of β-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.
Chymotrypsin and trypsin inhibitor isolated from potato tubers by T. A. Revina; I. A. Parfenov; E. L. Gvozdeva; N. G. Gerasimova; T. A. Valueva (pp. 239-244).
Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., cv. Zhukov’s Jubilee) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for subtilisin Carlsberg. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.
Purification of extracellular proteinases from B. subtilis SKB 256 by biospecific chromatography by U. R. Radzhabov; K. D. Davranov; M. M. Rakhimov (pp. 245-249).
A simple and efficient method of preparing highly purified extracellular proteinases of B. subtilis B-1 (SKB 256) has been developed. A sorbent based on sorsilen impregnated with hemoglobin or cytochrome c has been synthesized for this purpose. A significant difference between the efficiency of hemoglobin and cytochrome c as biospecific ligands has been observed: the enzyme yield amounted to 40.6 and 65.6% of the total amount of enzyme adsorbed, respectively. The culture was shown to contain two major proteinase forms with different molecular masses that could be separated by chromatography on a Sephadex G-50 but gave only one band with MW 27 kDa upon denaturing electrophoresis in 12.5% PAG in the presence of 0.1% SDS. The influence of eluent pH, ionic strength and ethanol concentration on the sorption of the proteinases on the biospecific sorbent, as well as on the desorption from it, has been investigated. Positive influence of 20% ethanol on proteinase desorption has been demonstrated.
Purification and characterization of an endoxylanase from the culture broth of Bacillus cereus BSA1 by A. Mandal; S. Kar; P. K. Das Mohapatra; C. Maity; B. R. Pati; K. C. Mondal (pp. 250-255).
An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K m and V max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu2+, Hg2+. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.
Rapid differentiation of bacterial species by high resolution melting curve analysis by J. Šimenc; U. Potočnik (pp. 256-263).
Molecular based differentiation of various bacterial species is important in phylogenetic studies, diagnostics and epidemiological surveillance, particularly where unusual phenotype makes the classical phenotypic identification of bacteria difficult. Molecular approach based on the sequence of 16S ribosomal RNA gene analysis can achieve fast and reliable identification of bacteria. High resolution melting (HRM) curve analysis has been developed as an attractive novel technique for DNA sequence discrimination but it’s application for bacteria differentiation has not been well studied yet. We have developed HRM assay for differentiation of sixteen pathogenic or opportunistic bacterial species. Amplified partial 16S ribosomal RNA gene region between 968 and 1401 positions (E. coli reference numbering) was subsequently used in high resolution melting curve analysis of PCR products for bacterial species differentiation. Sixteen bacterial species were simultaneously discerned by difference plot of normalized and temperatures shifted melting curves, without need for spiking of DNA, hetero-duplexing experiments or application of several primer pairs. High resolution melting curve analysis of duplex DNA is simple, fast and reliable tool for bacterial species differentiation and may efficiently complement phenotypic identification of bacteria.
Antistress cross-protection of UV-irradiated yeast cells with participation of extracellular peptide factors by L. I. Vorob’eva; E. Yu. Khodzhaev; M. M. Vustin (pp. 264-269).
Antistress effect of extracellular peptides on UV-irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast—Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.
Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis by V. A. Andryushina; N. V. Rodina; T. S. Stytsenko; Luu Duc Huy; A. V. Druzhinina; V. V. Yaderetz; N. E. Voishvillo (pp. 270-273).
Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5–15 μm) or the transformation in the presence of randomly methylated β-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The crude product ADD was obtained in 75% yield, based on phytosterol. It contained as by-products 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment—the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD—no by-products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5% AD was obtained. The yield of 9-OH-AD (m.p. 218–220°C) based on transformed phytosterol was 56%.
Effect of phytohormones synthesized by rhizosphere bacteria on plants by M. G. Sokolova; G. P. Akimova; O. B. Vaishlya (pp. 274-278).
New strains of rhizosphere microorganisms Azotobacter chroococcum Az d10, Bacillus megaterium Pl-04, and Bacillus mucilaginosus B-1574 were found to be able to synthesize cytokinins (CKs) and indolylacetic acid (IAA). Three forms of CKs—dihydrozeatin riboside, isopentenyl adenosine, and trans-zeatin riboside—were identified, whose ratio was different in the three bacterial cultures. Inoculation of cucumber (Cucumis sativus L.) plants increased the content of CKs and IAA in them by 35.6 and 21.3%, respectively, and also stimulated seed germination and increased the growth rate, the biomass of shoots, the number of lateral roots, and the root hair area, which ensured better plant nutrition. The IAA/CKs ratio shifted during bacterization towards CKs due to increase in the content of riboside forms, which apparently caused growth stimulation.
Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain by A. M. Rozhkova; A. S. Sereda; N. V. Tsurikova; A. K. Nurtaeva; M. V. Semenova; L. V. Rimareva; E. A. Rubtsova; I. N. Zorov; O. A. Sinitsyna; A. P. Sinitsyn (pp. 279-287).
A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of glucoamylase. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and Aspergillus niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6–14% range of total protein.
New producers of biologically active compounds—fungal strains of the genus Penicillium isolated from permafrost by T. V. Antipova; V. P. Zhelifonova; B. P. Baskunov; S. M. Ozerskaya; N. E. Ivanushkina; A. G. Kozlovsky (pp. 288-292).
Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazinees, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxyagroclavine-I and quinocitrinines. The main physiological and biochemical characteristics of this producer were investigated.
Effect of organic and inorganic toxic compounds on luminescence of luminous fungi by G. A. Vydryakova; A. A. Gusev; S. E. Medvedeva (pp. 293-297).
The possibility of the development of the solid phase bioluminescent biotest using aerial mycelium of luminous fungi was investigated. Effect of organic and inorganic toxic compounds (TC) at concentrations from 10−6 to 1 mg/ml on luminescence of aerial mycelia of four species of luminous fungi—Armillaria borealis (Culture Collection of the Institute of Forest, Siberian Branch, Russian Academy of Sciences), A. mellea, A. gallica, and Lampteromyces japonicus (Fungi Collection of the Botanical Institute, Russian Academy of Sciences)—has been studied. Culture of A. mellea was shown to be most sensitive to solutions of the model TC. It was demonstrated that the sensitivity of the luminous fungi is comparable with the sensitivity of the bacteria that are used for environmental monitoring. Use of the aerial mycelium of luminous fungi on the solid support as a test object is a promising approach in biotesting for the development of continuous biosensors for air monitoring.
Melanin of Laetiporus sulphureus (Bull.: Fr.) Murr sterile form by D. N. Olennikov; S. V. Agafonova; A. V. Stolbikova; A. V. Rokhin (pp. 298-303).
Melanin complex was isolated from mycelium of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr (with a yield of 2.49% of the dry weight). UV and IR spectroscopies, gel chromatography, and alkaline cleavage assay demonstrated that the isolated melanin was heterogeneous and belonged to the dihydronaphthalene type. 13C-NMR data suggested that aromatic fragments were dominant in the melanin structure. In vitro study of the antioxidant action demonstrated that the L. sulphureus melanin displayed a radical-scavenging activity and the ability to inactivate hydrogen peroxide and nitrogen(II) oxide molecules and to chelate iron(II) ions.
Bioengineering of symbiotic systems: Creation of new associative symbiosis with the use of lectins on the example of tobacco and oil seed rape by Z. R. Vershinina; An. Kh. Baimiev; D. K. Blagova; A. V. Knyazev; Al. Kh. Baimiev; A. V. Chemeris (pp. 304-310).
Hairy roots in tobacco and oil seed rape transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression of lectin gene on colonization of transgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and oil seed rape. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.
Biopolymer of alginate nature with a predominance of L-guluronic acid by Ya. O. Loginov; G. G. Khudaigulov; S. P. Chetverikov; A. I. Melent’ev; O. N. Loginov (pp. 311-314).
Highly viscous polysaccharide (250–350 kDa) of an alginate nature with a predominance of α-L-guluronic acid (M/G = 0.22) was obtained from Azotobacter vinelandi. The yield of polysaccharide was 20.5 ± 0.5 g/l when cultured in a medium containing molasses at a viscosity of the cultural liquid of over 30000 cSt. The biopolymer is stable at pH 4.0–9.0 in a wide temperature range and well soluble in highly mineralized water; it retains a high viscosity level and can be used in the petroleum industry for enhanced oil recovery.
Effect of soybean lipoxygenase on baking properties of wheat flour by M. D. Permyakova; V. A. Trufanov (pp. 315-320).
Changes in bread-baking properties of wheat flour caused by soybean lipoxygenase and polyunsaturated fatty acids were studied. A positive effect of soybean flour added during dough kneading in an amount of 2% was demonstrated. A method for dough fermentation increasing the loaf volume and improving organoleptic characteristics and total bread-baking estimate is recommended.
Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk by M. A. Burkin; I. A. Galvidis (pp. 321-326).
As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.
Trotsenko, Yu.A., Doronona, N.V., and Torgonskaya, M.L. Aerobic Methylobacteria. Pushchino: ONTI PSC RAS, 2010
by V. I. Tishkov (pp. 327-327).