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Applied Biochemistry and Microbiology (v.45, #5)
Steroid furostanol glycosides: A new class of natural adaptogenes (Review) by I. S. Vasil’eva; Zh. V. Udalova; S. V. Zinov’eva; V. A. Paseshnichenko (pp. 463-472).
The present review summarizes experimental data revealed while studying the mechanism of the adaptogenic effect of furostanol glycosides (FG) extracted from Dioscorea deltoidea Wall cell culture under the conditions of biotic stress in tomato plants Lycopersicon esculenium Mill. induced by the gall nematode Meloidogyne incognita Kofoid et White. Comparison of changes in isoprene content (phytosterines, tomatin, and carotenoids) and in the rate of oxidative processes in the leaves and roots of intact and treated plants evidence that FG cause nonspecific defense reactions resulting in the formation of systemic acquired resistance. This formation is presented by the enhancement in photosynthetic apparatus pigment fund, pigments of the violaxanthin cycle in particular, by activation of processes related to POL, and by increase in peroxidase activity—enzyme of antioxidant protection.
Thermoregulation of catalytic activity of enzymes polymeric derivatives by I. L. Valuev; L. V. Vanchugova; Yu. A. Taluzhenkov; L. I. Valuev; T. A. Valueva (pp. 473-477).
Trypsin was immobilized on polymeric carriers with low critical solution temperature (LCST). Homopolymer of N,N-diethylacrylamide (DEAA), random copolymers of DEAA and acrylamide (AA), and block copolymers poly-DEAA-poly-AA were used as the carriers. It was shown that at a temperature above LCST all carriers have a conformation change and trypsin’s polymeric derivatives precipitate. The maximal activity after phase transition keeps trypsin, immobilized on poly-DEAA block in poly-DEAA-poly-AA block-copolymer.
Immobilization of Alcaligenes faecalis penicillin G acylase on epoxy-type supports by J. Sun; Y. Zhou; Z. Yuan; G. Xu (pp. 478-483).
Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.
The study of two alkaliphilic thermophile bacteria of the Anoxybacillus genus as producers of extracellular proteinase by E. V. Lavrenteva; A. P. Shagzhina; O. B. Babasanova; Y. E. Dunaevsky; Z. B. Namsaraev; D. D. Barkhutova (pp. 484-488).
Two strains of alkaliphilic thermophile bacteria of the genus Anoxybacillus from hydrothermal vents of Lake Baikal were detected and characterized. It was demonstrated that proteinases secreted by these bacteria had wide substrate specificity, hydrolyzed proteins and n-nitroanilide substrates, and showed maximal activity at pyroglutamyl-alanine-alanine-leucine n-nitroanilide hydrolysis. We determined maximal activity of the proteinases at alkaline pH values (10.0–10.5), the enzymes were thermostable and were characterized by a wide thermal optimum (55–70°C). The results of inhibitor analysis and substrate specifity examination of extracellular enzymes demonstrated their belonging to the subtilisin-like serine proteinases.
The mechanism of action of reactivating factor from Luteococcus japonicus subsp. casei by L. I. Vorob’eva; E. Yu. Khodzhaev; A. L. Mulyukin; I. Yu. Toropygin (pp. 489-493).
Reactivating factor (RF) from Luteococcus japonicus subsp. casei was shown to be constitutively synthesized and to act a by one-step mechanism, being activated independently from stress. Cell reactivation (reversion of a cell’s ability to form macrocolonies) might be ensured by the membrane mechanism of RF action, which is proved with the dependence of antistress activity from the condition of the cytoplasmic membrane and with the form of concentration dependence. The incubation of UV-treated L. casei suspension with RF increased the number of cells with intact barrier membrane (1.6–1.8-fold increase compared to RF-untreated cells) and the number of colony-forming cells. Cross defensive and reactivating RF effects on both L. casei and yeast Saccharomyces cerevisiae cells were described. Bacterial and yeast’s RF compete for membrane receptors. Matrix Assisted Laser Desorption/Ionization time-of-flight (MALDI-TOF) spectrometry revealed that RF of L. casei contained two major peptides of 5.8 and 7.6 kDa, while RF of S. cerevisiae was represented by a single peptide of 5.8 kDa. The presence of 5.8 kDa peptide in RF from bacteria and yeasts might ensure cross responses in these organisms.
Amine neuromediators, their precursors, and oxidation products in the culture of Escherichia coli K-12 by V. A. Shishov; T. A. Kirovskaya; V. S. Kudrin; A. V. Oleskin (pp. 494-497).
The growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains (i) the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and (ii) the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.
Metabolism characteristics of Chelativorans oligotrophicus by two-phase growth on the mixture of EDTA and glucose by E. N. Kaparullina; N. V. Doronina; L. V. Trilisenko; V. M. Vagabov; Yu. A. Trotsenko (pp. 498-502).
The obligate destructor of ethylene diamine tetraacetate—a culture of Chelativorans oligotrophicus LPM-4—did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase) were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates in glucose metabolism as a supplementary energy source.
2-14C-methylerythritol 2,4-cyclodiphosphate incorporation into bacterial and plant isoprenoids by K. V. Mazikin; Yu. V. Ershov; A. V. Goncharenko; D. N. Ostrovskii (pp. 503-505).
To work out a test system for studying the inhibition of isoprenoid biosynthesis through non-mevalonate pathway, the conversion of 2-14C-methylerythritol 2,4-cyclodiphosphate ( 14 C-MEC) into isoprenoids of three bacteria species and into plastids of 11 higher plant species was studied. The highest efficiency (up to 63%) of the process was observed with chromoplasts from narcissus (Narcissus pseudonarcissus L.) and celandine (Chelidonium majus L.). Twenty five percent of added 14C-MEC was recovered in an isoprenoid fraction of red pepper (Capsicum annuum L.) chromoplasts. Thus, these three models can be used to test antibiotic inhibitors of isoprenoid biosynthesis.
Complex formation between trigliceropeptides pseudomonades and plant root exudates as a mechanism of action on phytopathogens by S. P. Chetverikov; L. R. Suleimanova; O. N. Loginov (pp. 506-510).
The influence of plant exudates on the antifungal activity of Pseudomonas chlororaphis strains (IB 6, IB 51) and P. putida (IB 17) was investigated in model experiments. Using UV spectroscopy and polarimetry methods, triglyceropeptide metabolites from the Pseudomonas strain were shown to form intermolecular complexes with plant exudates such as carbohydrates, organic acids, and amino acids. The stoichiometric contents of metabolite-exudate complexes were evaluated.
Synergistic action of entomopathogenic hyphomycetes and the bacteria Bacillus thuringiensis ssp. morrisoni in the infection of Colorado potato beetle Leptinotarsa decemlineata by V. Yu. Kryukov; V. P. Khodyrev; O. N. Yaroslavtseva; A. S. Kamenova; B. A. Duisembekov; V. V. Glupov (pp. 511-516).
A synchronous coinfection of the Colorado potato beetle Leptinotarsa decemlineata (Say) with the entomopathogenic bacteria Bacillus thuringiensis ssp. morrisoni Bonnifoi & de Barjak var. tenebrionis Krieg et al. and hyphomycete Metarhizium anisopliae (Metsch.) Sorokin or Beauveria bassiana (Bals.) Vuill leads to the rapid death of 95–100% of larvae. The bacteria arrest the nutrition of insects, while the fungal spores kill the weakened larvae. The synergistic effect of two pathogens is recorded at a relatively low hyphomycete titer (1–5 × 106 conidia/ml) and is evident in the mortality dynamics at all larval ages. These bacterial and fungal pathogens display no antagonism on artificial nutrient media. This microbial complex is highly efficient under natural conditions (80–90% larval mortality rate and no plant defoliation).
Characteristics of the succinate transport into Saccharomices cerevisiae cellsafter prolonged cold preincubation by D. A. Aliverdieva; D. V. Mamaev; L. S. Lagutina (pp. 517-524).
A nonconventional approach to the measurement of succinate transport through plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport through plasmalemma. Impermeable specific inhibitor of plasmalemma dicarboxylate transporter was employed as a tool to optimize conditions for the transport activity assay. For this purpose yeast culture was grown in synthetic medium. We selected conditions empirically. After aerobic preincubation of S. cerevisiae cells at 0°C (instead of incubation at 15°C), the rate of endogenous respiration decreased substantially and was stabilized during measurements at a level that was five times lower than oxidation rates in the presence of exogenous substrate. Linearity of Dixon plots for succinate oxidation depression by impermeable O-palmitoyl-L-malate is a test for selected conditions of measurement of plasmalemmal succinate transport. This approach allowed for the reproducible determination of K m for the dicarboxylate transporter (7.3 ± 2.1 mM) within a half-hour period. The advantages and drawbacks of this fast, but indirect, assay of slow substrates transport into the cell are compared with conventional methods.
Construction of flocculent industrial yeast by the yeast flocculation gene FLO1 by F. Z. Wang (pp. 525-530).
The structure gene FLO1 from Saccharomyces cerevisiae W303-1A encoding a flocculation protein and the G418 resistance gene kanMX from plasmid pUG6 were amplified by PCR method. The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Angel yeast. The transformant Angel yeast F6 was obtained and showed strong and stable flocculation ability during 20 batches inoculation. And the flocculation ability of the transformant Angel yeast F6 showed no difference in the medium with the initial pH ranging from 3.5 to 6.0. Noteworthily, the flocculation onset of the transformant strain was in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And in the experiment the ethanol yield and other properties of the transformant Angel yeast F6 were similar to those of the wild-type strain, although its fermentation time was a little slower comparing with the wild-type strain. Those would be potential application for yeast cells to separate and recycle in the fuel ethanol industry.
Carbon and nitrogen source effects on basidiomycetes exopolysaccharide production by V. I. Elisashvili; E. T. Kachlishvili; S. P. Wasser (pp. 531-535).
The capability to synthesize the extracellular polysaccharide (EPS) is widespread among eight mushroom species which accumulated 0.6–2.2 g/1 of EPS in submerged cultivation. Glucose, maltose, and mannitol were the most appropriate carbon sources for biomass and EPS production. Organic nitrogen sources appeared to be the most suitable nitrogen sources for biomass and EPS accumulation. The cultivation process in shake flasks was successfully reproduced in a laboratory fermentor with enhanced EPS production. The highest yield of EPS (3.8–4.0 g/1) was achieved in cultivation of Agaricus nevoi and Inonotus levis.
Water-soluble endopolysaccharides from the fruiting bodies of Laetiporus sulphureus (Bull.:Fr.) Murr. by D. N. Olennikov; S. V. Agafonova; G. B. Borovskii; T. A. Penzina; A. V. Rokhin (pp. 536-543).
Endopolysaccharides represented by glucans, galactans, and glycoproteins were isolated from the fruiting bodies of the sulfur shelf (Laetiporus sulphureus (Bull.:Fr.) Murr.), which were obtained by the naturalplantation method. The study of the major 56-kDa polysaccharide, laetiporan A, the content of which accounted for 0.28% of the fruiting body weight, showed that it is a β-1,3-glucan containing mannose, galactose, fucose, xylose, and rhamnose residues at position C-6. An antioxidant effect of laetiporan A was discovered.
The composition of volatile components of dry cepe and oyster mushroom by T. A. Misharina; S. M. Mukhutdinova; G. G. Zharikova; M. B. Terenina; N. I. Krikunova; I. B. Medvedeva (pp. 544-549).
The composition of aroma compounds in dry cepe mushroom (Boletis edulis Fr.) and oyster mushroom (Pleurotus ostreatus Fr.) was studied using capillary gas chromatography and chromatography—mass spectrometry. In dry cepe, 53 volatile compounds were identified, and in dry oyster mushroom 41 compounds were identified. Volatile organic substances with various functional groups formed the flavor of dry mushrooms. Unsaturated alcohols and ketones with eight carbon atoms were responsible for the mushroom notes of products. Their content in dry cepe was much higher than in dry oyster mushroom. The specific aroma of dry cepe was formed by the complex mixture of methional, substituted furans, pyrazines, and pyrroles. The content of these compounds was higher in dry cepe than in dry oyster mushroom. The content of aromatic and aliphatic aldehydes with six, nine, and ten carbon atoms was higher in dry oyster mushroom. The differences in the qualitative and quantitative composition of volatile compounds are responsible for more intensive and pleasant aroma of dry cepe in comparison to that of dry oyster mushroom.
The impact of melaphene on the expression of chloroplastic chaperone protein HSP70B and photosynthetic pigments in cells of Chlamydomonas reinhardtii by O. V. Ermokhina; G. G. Belkina; Yu. P. Oleskina; S. G. Fattakhov; N. P. Yurina (pp. 550-554).
The effects of growth regulator of the new generation—melamine salt of bis(oxymethyl)phosphine acid (melaphene)—on culture growth, pigment and protein content, and the induction of protective chloroplastic chaperone HSP70B in Chlamydomonas reinhardtii CW15 cells were studied. Melaphene exhibited 10–30% growth inhibition at 10−9–10−2% concentration. At 10−9–10−4% of melaphene electrophoretic concentration, the pattern of cellular proteins was similar to the control. The alterations in protein content of algae cells were detected only at 10−2% concentration. The content of chlorophyll and carotenoids in melaphene-treated cells was 17–40% lower than in the control. Melaphene at 10−9–10−2% concentration inhibited HSP70B induction by 39–43% compared to untreated cells. The potential mechanism of melaphene effect might involve its influence on nuclear gene expression.
Stability of the rolC gene and its expression in 15-year-old cell cultures of Panax ginseng by K. V. Kiselev; V. P. Bulgakov (pp. 555-561).
After 15 years of cultivation of Panax ginseng, transgenic cell cultures were shown to express rolC gene at a high level. We determined that the rolC gene underwent mutagenesis. In particular, from one to four nucleotides were changed for the whole gene sequence (540 bp). These substitutions were synonymous in half of the cases or resulted in the modification of single amino acids in the rolC-encoded protein. With the example of β-1,3-glucanases we showed that long cultivation and the observed changes in nucleotide sequences of the transgene did not inhibit the activating effect of rolC on enzymatic activity of β-1,3-glucanases.