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Applied Biochemistry and Microbiology (v.36, #4)
Production and purification of protein hydrolysates (review) by A. D. Neklyudov; A. N. Ivankin; A. V. Berdutina (pp. 317-324).
Recent achievements in the technology of producing protein hydrolysates are reviewed. Approaches to describing the mechanism of hydrolysis and the possibility of purifying hydrolysates for their use in the food, medical, forage, and microbiological industries are discussed.
Use of membraneless osmosis for concentration of proteins from molecular-dispersed and colloidal-dispersed solutions (review) by Yu. A. Antonov (pp. 325-337).
Theoretical principles, practical realizations, and future trends in the use of the method of protein concentration based on thermodynamic incompatibility of proteins with polysaccharides are reviewed. The relationship between structural features of these biopolymers (molecular weight, rigidity, and conformation of the polysaccharide chain; the nature and ionogenic properties of its functional groups; the type of protein and state of its molecules), as well as major physicochemical parameters (pH, ionic strength, and temperature) and mechanical shift energy of the system, on the one hand, and its phase diagram, on the other hand, are discussed.
New cyclomaltodextrin glucan transferases produced by bacillus macerans by V. A. Abelyan; K. B. Afyan; L. S. Manukyan (pp. 338-343).
—For the first time, microorganisms producing cyclomaltodextrin glucantransferaseglucan transferases (CGT, EC 2.4.1.19) were isolated from soil samples of various ecogeographical regions. These microorganisms were identified asBacillus macerans. The enzymes were purified by affinity chromatography on an α-cyclodextrin polymer and gel filtration on Biogel P-150 and proved to be electrophoretically homogeneous. Some of their physicochemical and biochemical properties are reported.
Isolation and characterization of extracellular lipase preparations from wild-type (V-10) and mutant (M-1)Serratia marcescens strains by A. B. Duzhak; Z. I. Panfilova; E. A. Vasyunina (pp. 344-352).
—The cultivation conditions of wild-type strain V-10 and mutant strain M-l (overproducer of endonuclease and chitinase) ofSerratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximum lipase yield (840 AU/ml) after 10–12 h of cultivation; the strain M-l (330 AU/ml), after 25–30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acidic medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed the highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and were inactive in the presence of cetyltrimethylammonium bromide.
Isolation and characterization of a micromycete, a producer of neutral catechol oxidases, from tropical soils with elevated dioxine content by L. G. Vasil’chenko; O. V. Koroleva; E. V. Stepanova; E. O. Landesman; M. L. Rabinovich (pp. 353-362).
—Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with an elevated phenol oxidase activity. As a result, a fast-growing nonsporulating strain producing neutral phenol oxidases was isolated and identified asMycelia sterilia INBI2-26. The strain formed extracellular phenol oxidases during surface growth on a liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of the culture liquid revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by Ultrafiltration, ion exchange chromatography, and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40°C., they retained at least 50% of activity after incubation for 50 h. At 50°C., PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3–6 h. The pH-optimutns for PO1 and PO2 toward catechol were 6 and 6.5; and theK m values were 1.5±0.35 and 1.25±0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 withK m values 1.6±0.18 and 0.045±0.01 mM, respectively, but displayed no activity toward tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.
Comparison of proteolytic activities of the enzyme complex from mammalian pancreas and pancreatin by A. V. Berdutina; A. D. Neklyudov; A. I. Ivankin; B. S. Karpo; S. I. Mitaleva (pp. 363-367).
The proteolytic activity and thermal stability of the enzyme complex of a cell suspension from pig and bovine pancreas glands was compared with those of pancreatin. The enzyme complex displayed the highest thermal stability and activity at 50°C. The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 ± 0.1) at which this complex had the maximum proteolytic activity were determined. Pancreatin had a pH optimum of 8.0 ±0.1.
Production of antibodies to roridin and their applications by A. A. Burkin; G. P. Kononenko; V. G. Zoryan; N. A. Soboleva; E. V. Zotova (pp. 368-371).
Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues—roridin A, verrucarin A, and verrucarol—amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 μg/kg.
Chitin and chitosan derivatives as elicitors of potato resistance to late blight by N. I. Vasyukova; G. I. Chalenko; N. G. Gerasimova; E. A. Perekhod; O. L. Ozeretskovskaya; A. V. Il’ina; V. P. Varlamov; A. I. Albulov (pp. 372-376).
Water-soluble low-molecular-weight (3–10 kDa) chitosan obtained by enzymatic degradation of high-molecular-weight chitosan, as well as its deaminated derivatives, can be used as elicitors of resistance to late blight in potato.
Melanin complex of the fungusInonotus obliquus by V. G. Babitskaya; V. V. Shcherba; N. V. Lkonnikova (pp. 377-381).
The fungusInonotus obliquus (Pers.) Pil. synthesized high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was shown that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated melanogenesis. The production of melanin correlated with the synthesis ofo- andp-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.
Partitioning of divalent metal ions in aqueous casein-containing systems of variable mineral compositions by N. K. Genkina; G. I. Koltysheva; M. N. Manakov (pp. 382-384).
The influence of the salt composition of casein-water-Cd(NO3)2 and defatted milk-Cd(NO3)2 systems on the partitioning of Cd2+ during acidic precipitation of casein was studied. The conditions that minimize the content of the toxicant ion in the isolated protein (pH and salt composition) were determined.
Effect of the composition of the medium on the characteristics of polydisulfide bioantioxidants by A. N. Eremin (pp. 385-393).
Characteristics of polydisulfides of gallic acid (PDSG), 2-amino-4-nitrophenol (PDSANP), and biuret (PDSB) depending on the composition of the aqueous medium were studied. In contrast to PDSANP and PDSB, there was oxidation of PDSG with accumulation of products of polydisulfide transformation in the medium. The rate of PDSG autoxidation depended on pH on the concentration of polydisulfide and buffers and increased at high pH. The rate of oxidation significantly increased in the presence of DMPA, ethanol, or CTAB (surfactant). Decreasing the pH of the solution and adding ovalbumin and/or Triton X-100 to the medium can decrease the rate of autoxidation of PDSG in an aqueous medium. Exogenous H2O2 inhibited the oxidation of PDSG. The secondary structure of catalase was changed by PDSG. Electrical conductance of PDSG and PDSANP solutions was studied. Possible mechanisms of PDSG autoxidation and polydisulfides-protein interaction due to forces of cooperative electrostatic interaction, thiol-disulfide exchanges, and nucleophilic replacements were discussed.
ß- Glucosidases from leaves and roots of the common beet,Beta vulgaris by N. S. Zakharova; T. A. Petrova (pp. 394-397).
ß-Glucosidases detected in the leaves and roots of the common beet,Beta vulgaris, have been demonstrated to catalyze hydrolysis of native betacyanins. A method is described for the isolation and purification of ßglucosidase from the roots, which involves ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex gel filtration. Maximum activity of the enzyme is detected at 50°C and pH 8.0; it retains stability within the pH range from 5.1 to 9.2. In leaves, ß-glucosidase is associated with chloroplast membranes; solubilization of the membranes results in enzyme inactivation.
Differential analysis of major natural cytokinins by enzyme immunoassay by A. N. Blintsov; M. A. Gusakovskaya; I. P. Ermakov (pp. 398-403).
An immunochemical method for quantitative differential analysis of major types of natural cytokinins in plant tissues subjected to minimal treatment, without purification or chemical modification of hormones, is proposed. This method is recommended for use in biology, medicine, and agriculture for determination of low-molecular-weight compounds having similar chemical structures but various biological activities.
Bacterial bioluminescence inhibition by Chlorophenols by A. D. Ismailov; S. I. Pogosyan; T. I. Mitrofanova; N. S. Egorov; A. I. Netrusov (pp. 404-408).
Photobacteria were used as a test object for rapid monitoring of ecotoxicants. Specific inhibitory effects of phenol and its chlorinated derivatives (2-chlorophenol, 2,3-dichlorophenol, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid) on bioluminescence and respiration of intact cells, as well as on the emission activity of the bioluminescence system and luciferase itself, were studied. The toxic effect on the photobacterial cells was found to increase as the number of chlorine atoms in the chlorophenol molecule increased. However, this trend was not observed in cell-free systems (purified luciferase or the protein fraction of a cell-free extract treated with (NH4)2SO4 at 40–75% saturation). Bacterial cells have a higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme system or luciferase. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechanism of inhibition with this substrate is typical of phenoxyacetic acids. With respect to FMNH2, all chlorophenols tested in this work were uncompetitive inhibitors. Oxygen uptake by photobacteria was shown to be insensitive to chlorophenols, at least within the concentration range that was effective in bioluminescence inhibition. The results of this study suggest that the bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria.
Effects of quinones on NADH-dependent enzymatic bioluminescent systems by N. S. Kudryasheva; E. N. Esimbekova; I. Yu. Kudinova; V. A. Kratasyuk; D. I. Stom (pp. 409-413).
The effects of a number of quinones on the bioluminescence characteristics of a three-component enzymatic system containing alcohol dehydrogenase, bacterial luciferase, and NADH-FMN oxidoreductase were studied to find the most sensitive kinetic parameters of the system intended to be used in biological testing. Both direct and back reactions catalyzed by alcohol dehydrogenase were studied in the presence and in the absence of quinones. The kinetic parameters of the bioluminescent system were found to depend on the redox potentials and concentrations of quinones. The quinone-induced effects were shown to be associated with changes in the NAD+/NADH ratio in the chain of NADH-dependent enzymes. The three-enzyme system based on alcohol dehydrogenase is suggested as a bioluminescence test for ecological monitoring of waste water.
Effects ofBacillus intermedius ribonuclease on the properties ofSaccharomyces cerevisiae by A. I. Kolpakov; F. G. Kupriyanova-Ashina; I. B. Leshchinskaya (pp. 414-417).
Effects ofBacillus intermedius ribonuclease on the physiological, biochemical, and consumer properties of baker’s yeastSaccharomyces cerevisiae were studied. This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors. The antistress effect of RNase correlated ith an earlier start of the stationary growth phase and increased trehalose pool.
Granular preparations ofAzotobacter containing clay minerals by I. K. Kurdish; L. V. Titova (pp. 418-420).
Interaction ofAzotobacter chroococcum 20 with clay minerals increased their cell viability at supraoptimal temperatures. Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage. The titers of viable bacteria in the preparations remained 60–70% of the initial level after 12 months of storage.
Proteolytic activity of viper venoms by S. V. Murzaeva; A. L. Malenev; A. G. Bakiev (pp. 421-424).
Proteolytic activities of venoms of vipers kept in a serpentarium for three years or captured in various environmental regions were estimated. Gurza venom contained considerable amounts of protein (830-930 μg/mg venom) and displayed a high proteolytic activity by tyrosine (80-140 μg/m in mg protein). The proteolytic activity did not depend on season or the age or physiological state of snakes in the reproductive period. The proteolytic activity of venom in gurza offspring was similar to that in parent specimens. Proteolytic activities (by tyrosine) of venoms produced by Radde’s vipers and common vipers were 77–90 and 18–36 μg/m in mg protein, respectively. The proteolytic activity of venom in common vipers native to the north European part of Russia was 20–30% higher than that in common vipers inhabiting southern European Russia. An inhibition assay found various ratios of metalloendopeptidase and serine endopeptidase activities in venoms of gurza, Radde’s viper, and common viper.
Activity of a new antimicrobial preparation from fish oils from various sources: Effects of different factors by V. G. Rybin; L. V. Shul’gina; D. V. Kuklev; T. M. Byval’tseva; Yu. G. Blinov; T. A. Davletshina; V. N. Akulin (pp. 425-428).
The induction of antimicrobial activity of a new preparation, an aqueous fraction of water-oil emulsion oxidized by atmospheric oxygen, was studied. The effect of varions factors (the degree of unsaturation of the initial oil and the content of oil oxidation products in the preparation) on antimicrobial activity was determined. The antimicrobial activity of the preparation was induced by oil oxidation. The preparation produced from sardineSardinops melanostica oil (33.95% polyunsaturated fatty acids) displayed the highest antimicrobial activity. Antimicrobial activity was shown in water-soluble oil oxidation products.
A portable reflectometric photometer for quantitative enzyme immunoassay by Yu. L. Shishkin; A. V. Zherdev; B. B. Dzantiev; Yu. A. Zolotov (pp. 429-433).
A portable optoelectronic reflectometric photocolorimeter was designed. The photocolorimeter implements two operating modes (transmission and reflection), and its performance was tested in two systems of enzyme immunoassay for testosterone: microplate ELISA and membrane dot-ELISA. The detection threshold for microplate ELISA and membrane dot-ELISA was 0.1 and 0.6 ng/ml, respectively. The coefficient of correlation between the photocolorimeter readings and conventional photometric methods is r=0.999. Relative standard deviation of the results of photocolorimetric measurements (n = 4) within the optical density range of 0.03 to 1.00 ranged from 3.4 to 0.7%. The simple and versatile design of the photocolorimeter makes it appropriate for testing various substances both in laboratory settings and in the field.
Changes in the lipid component of dried doughnut mixtures during storage by L. N. Shishkina; M. A. Klimova; G. F. Dremucheva; S. E. Traubenberg (pp. 434-438).
Changes in various indices of lipid components of dried doughnut mixtures during their joint or separate 3-month storage were studied. Methyl oleate oxidation model was used to determine the prooxidant properties of the initial mixture. The antioxidant activity, inhibition of oxidation, and inhibitory properties of lipids were shown to be enhanced during storage. The composition of phospholipids varied considerably due to changes in the degree of unsaturation of lipids in the mixture during storage. Joint storage of components resulted in greater stability and better preservation of lipid components (as compared to their separate storage).
Third international meeting “Biotechnologies-99” — presentation of innovative research and design project
by T.A. Reshetilova (pp. 439-442).