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Applied Biochemistry and Microbiology (v.41, #6)
Ultrasonic and Thermal Inactivation of Catalases from Bovine Liver, the Methylotrophic Yeast Pichia pastoris, and the Fungus Penicillium piceum by M. V. Potapovich; A. N. Eryomin; D. I. Metelitza (pp. 529-537).
The kinetics of inactivation of catalases from bovine liver (CAT), the fungus Penicillium piceum (CAT1), and the methylotrophic yeast Pichia pastoris (CAT2) was studied in phosphate buffer (pH 5.5 or 7.4) at 45 and 50°C or under the conditions of exposure to low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm2). The processes were characterized by effective first-order rate constants (s−1): k in (total inactivation), k in * (thermal inactivation), and k in * (us) (ultrasonic inactivation). The values of k in and k in * increased in the following order: CAT1 < CAT < CAT2. Circular dichroic spectra of the enzyme solutions were recorded in the course of inactivation by high temperatures (45 and 50°C ) and LFUS, and the contents of secondary structures were calculated. Processes of thermal and ultrasonic inactivation of catalases were associated with a decrease in the content of α helices and an increase in that of antiparallel β structures and irregular regions (CAT1 < CAT < CAT2). We conclude that the enzymes exhibit the following rank order of resistance: CAT1 > CAT > CAT2. Judging from the characteristics of CAT1, it appears to be an optimum component for antioxidant enzyme complexes.
Trypsin-Like Proteinases and Trypsin Inhibitors in Fruiting Bodies of Higher Fungi by L. A. Gzogyan; M. T. Proskuryakov; E. V. Ievleva; T. A. Valueva (pp. 538-541).
The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).
Isolation of Chitin-Specific Wheat Oxidoreductases by I. V. Maksimov; E. A. Cherepanova; L. G. Yarullina; I. E. Akhmetova (pp. 542-546).
Anionic peroxidase (pI ∼ 3.5) and oxalate oxidase (pI ∼ 7.0) were isolated from wheat seedlings using chitin. The strength of binding of enzymes with chitin depended on the degree of its acetylation and the ionic strength of the buffer. It was assumed that the acetyl groups of chitin are involved in sorption of enzymes on this biopolymer. The ability of anionic peroxidase and oxalate oxidase for sorption on chitin allows this biopolymer to be used for isolation of these proteins from plants. Coadsorption of anionic peroxidase and oxalate oxidase on chitin suggests that these enzymes cooperate to ensure a defensive response of wheat against chitin-containing pathogens.
Hydrogen Peroxide Content and Catalase Activity on Inoculation with Root Nodule Bacteria of Pea Seedlings with Different Ability for Nodulation by G. G. Vasil'eva; A. K. Glyan'ko; N. V. Mironova (pp. 547-550).
Hydrogen peroxide (H2O2) content and catalase activity were studied in pea (Pisum sativum L.) seedlings with a normal (cultivar Marat) and disrupted (pea mutants) process of nodulation that were inoculated with the nitrogen-fixing bacterium Rhizobium leguminosarum strain CIAM 1026. Differences in hydrogen peroxide content and catalase activity in pea seedlings with different ability for nodulation that were inoculated with rhizobia were found. It was assumed that H2O2 and catalase are involved in defensive and regulatory mechanisms in the host plant.
Bioconversion of β-Sitosterol and Its Esters by Actinobacteria of the Genus Rhodococcus by I. B. Ivshina; V. V. Grishko; E. M. Nogovitsina; T. P. Kukina; G. A. Tolstikov (pp. 551-557).
The ability of pure cultures of Rhodococcus actinobacteria from the Ural Specialized Collection of Alkanotrophic Microorganisms (World Federation for Culture Collections accession number 768; http://www.ecology.psu.ru/iegmcol) to convert β-sitosterol (BSS) and its 3β-acylated derivatives was studied. Rhodococcus strains with pronounced cholesterol oxidase activity, capable of converting BSS to stigmat-4-ene-3-one in the reaction of cooxidation with n-hexadecane, were selected. The dependence of the activity of cholesterol oxidase of rhodococci on the length of the acyl group in BSS esters was studied. Conditions under which Rhodococcus cells convert BSS to 17β-hydroxyandrost-4-ene-3-one (testosterone), commonly used in pharmacology, were determined.
Development of an Oil-Degrading Biopreparation by Activation of Aboriginal Hydrocarbon-Oxidizing Microflora by E. V. Pleshakova; N. N. Pozdnyakova; O. V. Turkovskaya (pp. 558-562).
A method of activation of aboriginal hydrocarbon-oxidizing microorganisms for remediation of soil and water basins polluted with oil products was developed. The optimum composition of activating additives was found (g/l): mineral components, 10.0; oil, 5.0; and a synthetic detergent, 0.2. The resulting biopreparations increased the degree of purification by factors of 4–8 in soil and 18–24 in water when applied at a concentration of 107 cells/g(ml).
Inhibitory Effects of Phenolic Ecotoxicants on Photobacteria at Various pH Values by V. V. Kouts; Yu. M. Il'ina; A. D. Ismailov; A. I. Netrusov (pp. 563-569).
Kinetic characteristics of light emission by intact cells of photobacteria Photobacterium phosphoreum and Vibrio harveyi were studied (at pH 5.5, 7.0, and 8.0), as well as inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) (at the same pH values). The emission kinetics lacked a steady state, irrespective of pH. At pH 5.5, luminescence decayed exponentially in the 60-s range; at pH 7.0 and 8.0, a 5-min luminescence activation was observed. The respiratory activity of the cells decreased by more than an order of magnitude at pH 5.5 (compared to the levels observed at pH 7.0 and 8.0). The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differed by one to two orders of magnitude, depending on pH. Maximum cell sensitivity to these compounds appeared at pH 5.5; minimum sensitivity, at pH 8.0. The effect of 2,6-DMP was pH-independent. The inhibitory effect was determined by the hydrophobicity of the molecule and pK values of the toxicants. At all pH values, substrate-depleted cells of photobacteria were more sensitive to chlorophenolic compounds than cells supplied with energy.
Cell Aggregation in Cultures of Micrococcus luteus, Studied by Dynamic Light Scattering by S. A. Voloshin; A. S. Kaprelyants (pp. 570-573).
Cell aggregation was studied using the method of dynamic light scattering in the course of growth of Micrococcus luteus cultures in a liquid medium. The method detects particles ranging in size from 0.5 to 1000 µm in samples containing no more than 105 cells/ml. When grown in liquid media, M. luteus forms aggregates; during the lag phase, 80% of the cells are found in aggregates of 10–1000 µm, only minor amounts being represented by single cells. With the onset of exponential growth, the aggregates were decomposed and single cells became prevalent in the culture liquid. This observation confirms that the aggregation of the cells during the lag phase is prerequisite to the initiation of bacterial growth. The method may be used in biotechnology for monitoring the state of bacterial cultures.
Changes in Catechins during the Fermentation of Green Tea by Y. Y. Tu; H. L. Xia; N. Watanabe (pp. 574-577).
The dynamics of tea catechins and organic acids in fermented fluid and yeast cells were studied. The concentration of eight kinds of catechins in solution decreased by 29.6–47.6%, respectively; some catechins were absorbed and accumulated by yeast cells, but the amount in the cells was very low during the fermentation process. The investigation of the catechins resolved in four citrate buffers with a pH range of 2.6–5.6 over 18 h showed that most catechins were stable in buffer solutions of pH 4.6 and 5.6, while significant losses took place in solutions of pH 2.6 and 3.6. However, most catechins were released and recovered by adjusting the pH value to 5.6, which suggested that catechins in extremely acidic buffer solutions might reversibly combine each other or with other compounds.
Genetic Differentiation of the Sherry Yeasts Saccharomyces cerevisiae by E. S. Naumova; Yu. V. Ivannikova; G. I. Naumov (pp. 578-582).
Molecular and genetic studies of the yeast Saccharomyces cerevisiae isolated at distinct stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from primary winemaking yeasts according to several physiological and molecular markers. All sherry strains, regardless of the place and time of their isolation, carry a 24-bp deletion in the ITS1 region of ribosomal DNA, whereas the yeasts of primary winemaking lack this deletion. Molecular karyotypes of sherry yeasts from different populations were found to be very similar.
Decolorization of Alcohol Distillery Wastewater by Thermotolerant White Rot Fungi by P. Chairattanamanokorn; T. Imai; R. Kondo; M. Sekine; T. Higuchi; M. Ukita (pp. 583-588).
To determine a thermotolerant fungus strain for decolorization of alcohol distillery wastewater (ADW), 38 fungus strains were studied. Capacity for ligninolytic enzyme production was examined at 35 and 43C on agar media containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and MnCl2. At 43°C, four Pycnoporus coccineus strains showed a higher potential for ADW decolorization both on agar media and in liquid media. Immobilized mycelia on polyurethane foam removed about threefold more total phenol than did free mycelia under conditions of shaking at 43°C. Moreover, immobilized mycelia removed nearly 50% more color than did free mycelia.
Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk by Zh. V. Samsonova; O. S. Shchelokova; N. L. Ivanova; M. Yu. Rubtsova; A. M. Egorov (pp. 589-595).
An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).
Phenol Conjugation with Peptides and Final Transformations of Conjugates in English Ryegrass Seedlings by D. I. Chrikishvili; E. P. Lomidze; T. I. Mithaishvili (pp. 596-599).
[1-14C]Phenol transformation in English ryegrass (Lolium perenne L.) sterile seedlings was studied. The compound studied was assimilated by a plant through leaves as vapor. Phenol was bound with ryegrass low-molecular-weight peptides producing phenol-peptide conjugates. Conjugation with endogenous peptides is one of the main pathways of phenol detoxication in ryegrass. Nearly three-fifths of phenol assimilated is conjugated with low-molecular-weight peptides. After removal of the plant from the labeled phenol-containing atmosphere, the content of conjugation products gradually decreased, followed by excretion of labeled carbon dioxide. This fact indicates that the conjugates are destructed and the carbon atoms of their radioactive component are oxidized to carbon dioxide. Almost one-third of assimilated phenol is transformed via the oxidation pathway, but a small part of it is irreversibly bound with biopolymer molecules.
Response of Winter Wheat to Cold: Production of Phenolic Compounds and L-Phenylalanine Ammonia Lyase Activity by N. A. Olenichenko; N. V. Zagoskina (pp. 600-603).
The formation of soluble and polymeric (lignin) phenolic compounds, activity of L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and content of free L-phenylalanine during cold hardening of winter wheat plants (Triticum aestivum L.) were studied. Cold treatment increased accumulation of soluble phenolic compounds in leaves while not affecting the content of lignin. The opposite was observed in tillering nodes. The activity of PAL was lower than in control plants in both tissues, and the content of free L-phenylalanine in tissues increased.
Oxidase-Peroxidase Method of Ethanol Assay in Fermented Musts and Wine Products by N. M. Pavlishko; O. V. Ryabinina; T. A. Zhilyakova; I. Yu. Sakharov; V. G. Gerzhikova; M. V. Gonchar (pp. 604-609).
A new alcohol oxidase-peroxidase method of ethanol assay in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). The proposed method is less time- and labor-consuming and allows simultaneous analysis of a series of wine samples.
Antioxidant Properties of Essential Oils: Autoxidation of Essential Oils from Laurel and Fennel and of Their Mixtures with Essential Oil from Coriander by T. A. Misharina; A. N. Polshkov (pp. 610-618).
Changes in the composition of essential oils from the seeds of laurel (Laurus nobilis L.) and fennel (Foeniculum vulgare Mill., var. dulce Thelling) and their mixture with essential oil from coriander were studied by capillary gas-liquid chromatography during storage in the dark and in light. Under these conditions, essential oil of laurel retained its composition for 12 months. Essential oil of fennel was rapidly oxidized in light. However, the rate of its oxidation in the dark was lower. The major component of essential oil of fennel, trans-anethol, had a lower antioxidant activity than essential oil of coriander. The mixture of essential oils from laurel and coriander possessed antioxidant properties and strongly inhibited the oxidation of components of the fennel oil.
The All-Russia Symposium on Biotechnology of Microbes
by N. N. Kolotilova; A. I. Netrusov (pp. 619-620).