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Applied Biochemistry and Microbiology (v.44, #6)
Covalent immobilization of urease on polysiloxane matrices containing 3-aminopropyl and 3-mercaptopropyl groups by R. P. Pogorilyi; V. P. Goncharik; L. I. Kozhara; Yu. L. Zub (pp. 561-565).
A sol-gel method of covalent immobilization of urease on polysiloxane matrices is developed which uses glutaraldehyde or Ellman’s reagent as binding agents. We show that the urease covalently bound to the poly(3-mercaptopropyl)siloxane matrix retains 67–84% of its activity and is stable, losing only 10% of its activity after 300 days. The urease adsorbed on the poly(3-mercaptopropyl)siloxane matrix was more active than the native urease. On the basis of the literature, we suggest that, in this case, the 3-mercaptopropyl groups of the polysiloxane matrix are brought into close proximity to the active site of urease, where they possibly act as proton donors, which results in an accelerated enzymatic reaction. Covalent immobilization of urease on the polysiloxane matrix containing 3-aminopropyl was less efficient, because the immobilized enzyme was significantly less active. At the same time, the urease adsorbed on the same matrix showed high activity (60–86%).
Physiological and biochemical characteristics of an isomaltulose synthase producer Erwinia rhapontici by O. S. Korneeva; O. Yu. Bozhko; Z. M. Mangueva (pp. 566-570).
The following conditions of isomaltulose synthase synthesis by Erwinia rhapontici bacteria at submerged cultivation were optimized: cultivating temperature of 30°C, culturing media initial pH of 7.5, and cultivation for 54 h in the media containing 10% sucrose. Electrophoretically homogeneous preparation with specific activity of 210 U/mg of protein was obtained. Optimal conditions for enzyme functioning were 30°C at pH 6.0–7.0. The enzyme activity was 3300 U/ml, which is 40–50 times higher, than catalytic activity of any of the strains studied previously.
Specific structural features and immunomodulatory properties of the lipopolysaccharides of Pseudomonas bacteria by S. N. Veremeichenko; G. M. Zdorovenko (pp. 571-579).
The results of in vitro studies of the immunomodulatory action of the lipopolysaccharides (LPS) of the Pseudomonas bacteria—P. fluorescens biovar I strains IMV 4125 = ATCC 13525, IMV 7769, and IMV 1152; P. fluorescens biovar IV strain IMV 2111; P. syringae pv. syringae IMV 281 = CPPB 281 = ATCC 19310 and IMV 467; and P. wieringae IMV 7923-on the mouse spleenocytes and human peripheral blood mononuclear cells (PBMC), B lymphocytes, and T lymphocytes are described. The proliferative activity of mouse spleenocytes correlated with the degree of LPS toxicity. The PBMC mitogenic activity induced by the P. fluorescens IMV 7769 LPS preparation exceeded the activity of E. coli 026:B6 LPS. The immunomodulatory effect of LPS on T cells was strain and dose dependent. The LPS of P. syringae pv. syringae INV 467 displayed a comparatively pronounced immunomodulatory effect on human blood B lymphocytes.
Conversion of 17α-methyltestosterone to methandrostenolone by the bacterium pimelobacter simplex VKPM Ac-1632 with the presence of cyclodextrins by A. V. Druzhinina; V. A. Andryushina; T. S. Stytsenko; N. E. Voishvillo (pp. 580-584).
Conditions of conversion of 17α-methyltestosterone to methandrostenolone with the presence of modified β-cyclodextrins (methylcyclodextrin, hydroxypropylcyclodextrin, and hydroxyethylcyclodextrin) in the steroid: cyclodextrin ratio 1: 1 were studied. The experimental solutions of modified β-cyclodextrins were prepared in deionized water with 5–7% methanol. Under the conditions found to be optimal, 1,2–dehydrogenation of 17α-methyltestosterone was carried out with 2–4 g/l Pimelobacter simplex VKPM Ac-1632 biomass. At the substrate concentration 5–20 g/l, the reaction occurred for 1–15 h without any by-products. The maximum rate of methandrostenolone accumulation was observed with hydroxypropylcyclodextrin. The methylcyclodextrin solution can be reused for complete 17α-methyltestosterone conversion at the concentration 5 g/l.
Activation of indigenous micorflora in oil-contaminated soils using photoluminescent films by L. I. Svarovskaya; L. K. Altunina; D. A. Filatov (pp. 585-589).
A stimulating effect of sunlight transformed by a photoluminescent polymer film on the abundance dynamics and fermentation and respiration of indigenous microflora in oil-contaminated soils was investigated. Polymer film doped with photoluminophores based on inorganic Eu-complexes and common glasshouse film was used as a cover material for oil-contaminated soils at experimental and control sites. The application of photoluminescent film has been reported to stimulate a hundredfold growth of the microflora population, with the soil respiration intensity and catalase activity being increased by a factor of 2.5–3, respectively. The extents of biodegradation of petroleum hydrocarbons within 60 days were up to 70 and 30% of the overall background pollution level for the experimental and control site, respectively. Residual hydrocarbons extracted from samples of the contaminated soils were analyzed by infrared spectroscopy to show the appearance of additional absorption bands at 3350, 1600, and 1710 cm−1, thus indicating the formation of metabolites during enzymatic oxidation of oil. Chromatographic data corroborated the occurrence of intense oxidation. The hydrocarbon biodegradation factor increases sixfold when the photoluminescent films are used.
Use of cadmium hydroxide gel for isolation of extracellular catalases from Penicillium piceum and characterization of purified enzymes by A. N. Eremin; I. V. Moroz; R. V. Mikhailova (pp. 590-599).
We optimized the conditions for isolation of extracellular catalases from Penicillium piceum F-648 and P. piceum A3 by means of volume chromatography with cadmium hydroxide gel. Our study showed that 55–57 mg wet gel are sufficient for the maximum sorption of catalase from 1 ml of culture fluid. This gel was formed in 1 ml 70 mM Cd(NO3)2 after addition of NaOH (Cd(NO3)2/NaOH molar ratio 1: 2.2). The eluting solution contained 50 mM NaH2PO4(pH 7.0), 5.0 mM dithiothreitol, and 0.3% sodium cholate and was potent in desorbing catalase from the gel. Subsequent ultrafiltration of the eluate on the membrane with a retention limit of 50 kDa allowed us to concentrate and purify the sample from low-molecular-weight protein impurities. NH4Cl (1.0 M) containing 0.3% sodium cholate was used to wash the sample from low-molecular-weight aromatic metabolites. Purified catalases included 33–34% antiparallel β-structures and 9%-spirals. Under optimal conditions in the medium of 10 mM phosphate buffered saline (pH 7.0) at 30°C, catalases from P. piceum F-648 were characterized by the following parameters: K M, 158.8 mM; catalytic constant, 2.83 × 106 s−1; enzyme inactivation rate constant in H2O2 decomposition, 3.5 × 10−2 s−1; and constant of the interaction between catalase complex I and second molecule of H2O2, 1.8 × 107M−1 s−1.
Genetic transformation of the mycelium fungi Acremonium chrysogenum by A. A. Zhgun; M. A. Ivanova; A. G. Domracheva; M. I. Novak; M. A. Elidarov; K. G. Skryabin; Yu. E. Bartoshevich (pp. 600-607).
The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C no. 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (ZeocinTM) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses.
Effect of various factors on the biosynthesis of piscarinines, secondary metabolites of the fungus Penicillium piscarium Westling by V. P. Zhelifonova; A. Maier; A. G. Kozlovskii (pp. 608-612).
Piscarinines A and B were synthesized most actively during the surface cultivation of the fungus Penicillium piscarium in a complex medium (5.5 mg/l). Under conditions of submerged cultivation in a mineral medium, the yield of piscarinines was two times lower. An increase in the inoculum quantity of conidia treated with Tween-80 increased the culture productivity. The biosynthesis of the alkaloid was completely suppressed when mannitol was replaced with glucose or when zinc, iron, or copper ions were added to the culture medium. The metabolites were active against the prostate cancer cell line LNCAP (IC50 were 2.195 and 1.914 μg/ml for piscarinines A and B, respectively).
Importance of lipidic acyl chains in the process of biochemical thermal adaptation of Cunninghamella japonica mucoraceous fungus by E. P. Feofilova; L. S. Kuznetsova (pp. 613-618).
The temperature of C. japonica cultivation influences the lipid content and composition of acyl chains, especially the content of such polyunsaturated acids as linoleic and linolenic. Thermal adaptation is accompanied by the modulation of fatty acid isomeric composition and acyl chain length and, at low temperatures, promotes the appearance of fatty acids uncommon to the fungus, in particular, arachidonic acid. The changes occur on a background of significant alterations in the fungus metabolism (in glucose uptake, ATP content, economic coefficient value, etc.). In experiments on the inhibition of translation with cycloheximide, abrupt temperature change (supraoptimal to cold) did not lead to desaturase de novo synthesis, but rather stimulated the activity of the named enzymes, except for palmitoleoyl-CoA desaturase. In the process of temperature adaptation, polar lipid microviscosity modulating compounds influenced fatty acid acyl chain composition. Microviscosity differences between polar and neutral lipids and correlation to the degree of fatty acid unsaturation during temperature fluctuation were established.
The effect of copper ions on the production of laccase by the fungus Lentinus (Panus) tigrinus by V. V. Shutova; V. V. Revin; Yu. A. Myakushina (pp. 619-623).
The basidiomycete Lentinus tigrinus was cultured in media containing copper ions added at different growth stages. Copper ions at increased concentrations decelerated of the fungal biomass accumulation. The later Cu2+ ions were added, the better the fungal mycelium developed, and the toxic effect of Cu2+ was less pronounced. The maximum laccase activity (47 U/ml) was observed in the presence of 1.5–2.0 mM Cu2+ added on day 4 of cultivation.
Novel liposomal forms of antifungal antibiotics modified by amphiphilic polymers by I. A. Yamskov; A. N. Kuskov; K. K. Babievsky; B. B. Berezin; M. A. Krayukhina; N. A. Samoylova; V. E. Tikhonov; M. I. Shtilman (pp. 624-628).
A novel method was developed to incorporate macrocyclic polyene antibiotics, nystatin and amphotericin B, into liposomes prepared from the mixture of phosphatidylcholine and cholesterol (7: 3) or phosphatidylcholine, cholesterol, and cardiolipin (7: 3: 1). Membranes of the liposomes were modified using the amphiphilic polymer N-vinylpyrrolidone with the molecular mass (MM) of the polymer fragment of 4000 and a single terminal n-octadecyl group. The content of the antibiotic incorporated within such nanosize liposomal carriers can reach 17–22%. The obtained modified liposomes, 150–200 nm in size, were more stable during prolonged storage and more resistant to various destructive factors, such as destabilizing agents (Triton X-100, ethanol) and ultrasound. The liposomal preparations showed higher antifungal activity than non-immobilized antifungal antibiotics.
Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A2-2 and monogalactosyldiacylgycerol by I. A. Li; A. M. Popov; A. V. Tsybul’skii; N. M. Sanina; E. Ya. Kostetskii; O. D. Novikova; O. Yu. Portnyagina; A. V. Mazeika (pp. 629-634).
A novel antigen carrier has been formulated on the basis of a cucumarioside-A2-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.
Effect of melaphene on expression of Elip1 and Elip2 genes encoding chloroplast light-induced stress proteins in barley by O. V. Osipenkova; O. V. Ermokhina; G. G. Belkina; Yu. P. Oleskina; S. G. Fattakhov; N. P. Yurina (pp. 635-641).
The effect of melaphene, a plant growth regulator of a new generation, on the growth, pigment composition, and expression of nuclear genes Elip1 and Elip2 encoding chloroplast light-stress proteins in barley (Hordeum vulgare L) seedlings was studied. It is shown that the height of seedlings treated with melaphene at concentrations of 0.5 × 10−10 and 0.5 × 10−8 M increased by approximately 10 and 20%, respectively, as compared to the control. At high concentrations (10−5 and 10−3 M), melaphene had no effect on the growth of seedlings. The content of chlorophylls and carotenoids in chloroplasts barely differed from the control at all melaphene concentrations tested. Reverse transcription-polymerase chain reaction (RT-PCR) showed that melaphene did not influence the expression of the nuclear gene encoding the low-molecular-weight plastid stress protein ELIP1. At the same time, the expression of the nuclear gene encoding the high-molecular-weight light-inducible stress protein ELIP2 in the plants treated with elaphene at a concentration of 0.5 × 10−8 M, increased by ∼70 %. At higher concentrations, melaphene suppressed the Elip2 gene expression. Thus, melaphene affects the expression of the Elip2 gene, which is involved in the regulation of chlorophyll synthesis and chloroplast biogenesis, which, in turn, may lead to changes in the resistance of plants to light-induced stress.
Effect of jasmonic acid on Ca+2 transport through the plasmalemma of potato tuber cells by E. P. Ladyzhenskaya; N. P. Korableva (pp. 642-646).
The rate of Ca2+ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10−5–10−10 M jasmonic acid on the accumulation of Ca+2 in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca+2,Mg+2-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca+2,Mg+2-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.
Characteristics of the composition of caucasian blackberry (Rubus caucasicus L.) leaves as a raw material for tea production by R. G. Melkadze; N. Sh. Chikovani; E. Z. Kakhniashvili (pp. 647-651).
The composition of Caucasian blackberry (Rubus caucasicus L.) six-leaf shoot was studied. The weight of the stem reached 50% of the total weight shoot. The content of moisture, extractive substances, and phenolic compounds was minimal at the beginning and end of the vegetation season. Phenolic compounds were represented by catechins, leukoanthocyanidins, and flavonols. The most abundant phenolic compounds in all parts of the blackberry shoot were leukoanthocyanidins, which accounted for approximately 50% of all compounds of this class. Phenolic compounds accumulated most actively in July and August. The average content of free amino acids in the blackberry leaf during the vegetation season was 26.68 mg/g. Among the total free amino acids, eleven have been identified, five of which proved to be essential (His, Arg, Met, Leu, Val) and accounted for 40 % of the total amount of amino acids. The oxidability of acetone extract of the blackberry leaf was compared to the oxidability of total phenolic compounds and tea tannin. The tea product obtained from the blackberry leaf had good organoleptic parameters and a saturated extractive complex.
Study of alcohol-soluble lignin in cognac spirits by A. F. Pisarnitskii; K. A. Askenderov (pp. 652-656).
We studied the structure of alcohol-soluble lignin in cognac spirits from Spain (7-year aging) and Azerbaijan (15-year aging). Alcohol-soluble lignin was precipitated and refractionated by means of preparative high-performance liquid chromatography. Lignin was studied by the method of high-performance liquid chromatography and mass spectrometry. Lignin hydrolysis products were assayed by the method of gas-liquid chromatography and mass spectrometry. The structure of lignans was reconstructed from these fragments. Lignans from Spanish and Azeri alcohol were arbitrarily designated as sinapoconiferal triglycoside and glucoferulate flavone diglycoside, respectively.