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Applied Biochemistry and Microbiology (v.36, #2)
ATP- and polyphosphate-dependent bacterial NAD+ kinases by S. Yu. Filippovich; T. P. Afanas’eva; G. P. Bachurina; M. S. Kritskii (pp. 97-100).
Measurable levels of activity of NAD+ kinases of actinomycetesMicrococcus luteus andCoryne-bacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacteriumEscherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD+ kinase inC. ammoniagenes andE. coli; four forms were found inM. luteus. All isoforms of this enzyme found inC. ammoniagenes andM. luteus displayed NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate,M. luteus is the most promising NADP producer organism.
High-solids enzymatic hydrolysis of steam-exploded willow without prior water washing by A. Pristavka; P. A. Kodituvakky; Yu. P. Kozlov; G. Zacchi; I. V. Berezin; M. L. Rabinovich (pp. 101-108).
A laboratory reactor equipped with a screw press was used for the hydrolysis of steam-SO2-exploded willowSalix caprea by a composition ofTrichoderma reesei andAspergillus foetidus enzyme preparations at high substrate concentration. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude the initial washing of steam-pretreated plant raw material by large volumes of water, which was necessary to eliminate the inhibitory effect of explosion byproducts on enzymatic hydrolysis. The first procedure included enzymatic prehydrolysis of the substrate for 1 h; separation of sugar syrup containing 40–60 g/l glucose, 20–25 g/l xylose, and up to 10 g/l disaccharides, as well as up to 35% of the initial enzymatic activity; then addition of a diluted acetate buffer (pH 4.5); and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l of xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by the added enzymes over 24 h. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons for inhibition of enzymatic hydrolysis are discussed.
Isolation and purification of acetolactate synthase and acetolactate decarboxylase from the culture ofLactococcus lactis by Yu. S. Kisrieva; V. M. Serebrennikov; N. A. Zagustina; A. M. Bezborodov (pp. 109-114).
Enzymes catalyzing the synthesis and subsequent transformation of α-acetolactate (AcL)—acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)—were isolated and partially purified from the cells of lactic acid bacteriaLactococcus lactis ssp.lactis biovar.diacetylactis, strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358 300 U/mg protein (9% yield). The preparation of AcLDC., purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparentK m, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic and the affinity of the enzyme for its substrate was high (K m = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.
Soluble and immobilized phenol oxidase of the fungusMycelia sterilia IBR 35219/2: A comparative study by N. I. Mchedlishvili; G. N. Pruidze; N. T. Omiadze; R. V. Zukhbaya (pp. 115-118).
Phenol oxidase (EC 1.14.18.1) from the microscopic fungusMycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased enzyme stability. Both soluble and immobilized forms of phenol oxidase fromM. sterilia IBR 35 219/2 catalyze oxidative conversion of phenolic compounds of green tea extract.
Key role of the medium in methemalbumin-catalyzed peroxidation of aromatic free radical scavengers and antioxidants by O. B. Rus’; A. V. Puchkaev; D. I. Metelitsa (pp. 119-127).
Participation of the complexes of hemin and albumins (or delipidated albumins) in peroxidation of aromatic free radical scavengers and antioxidants was studied at varying hemin/albumin ratios. The radicalscavenging amines includedo-phenylenediamine (OPD) and tetramethylbenzidine (TMB); the antioxidants were gallic acid (GA) and GA polydisulfide (GAPD). Peroxidation reactions were carried out in buffered physiological saline (BPS) supplemented with 2% dimethylsulfoxide (DMSO), pH 7.4 (medium A), or in 40% aqueous dimethylformamide (DMF), pH 7.4 (medium B). In all systems involving methemalbumins, kinetic constants (kcat), Michaelis constants(K M), and the ratios thereof(k cat/KM) were determined for OPD oxidation in medium A and TMB oxidation in medium B. Oxidation of OPD, GA, and GAPD in medium A was characterized by a decrease in the catalytic activity of hemin after the formation of hemin-albumin complexes. Conversely, oxidation of TMB and OPD in medium B was distinguished by pronounced activation of hemin present within methemalbumins.
Melanin pigments from the fungiPaecilomyces variotii andAspergillus carbonarius by V. G. Babitskaya; V. V. Shcherba; T. V. Filimonova; E. A. Grigorchuk (pp. 128-133).
Pigments synthesized by the micromycetesPaecilomyces variotii andAspergillus carbonarius are true melanins. Copper ions and bicyclic phenolic compounds stimulated melaninogenesis, whereas benzotriazole inhibited this process. Precursors of melanin pigments were obtained and identified.p-Hydroxybenzoic acid was shown to be the main product of melanin degradation. Melanins of these fungi are concluded to belong to the dihydronaphthalene group.
Inactivation of polygalacturonase during growth ofSaccharomyces pastorianus in culture by N. I. Astapovich; N. E. Ryabaya (pp. 134-137).
The effect of exogenous glucose addition on polygalacturonase (EC 3.2.1.15) activity in the culture medium ofSaccharomyces pastorianus was studied. A rapid but transient decrease in the enzyme activity was observed after 9–12 h after adding glucose to the culture medium. This effect was not associated with protein degradation or modification of the spectrum of secreted proteins. Ethyl acetate appeared in the culture medium durine this period.
Furan compounds produced by heating of fermented high-sacchariferous media by A. F. Pisarnitskii; I. V. Ulezlo (pp. 138-141).
5-Methylfurfural diethyl acetal, 5-methylfuran acid ester, and 4-hydroxypentanoic acid ethyl ester were first identified in high-sacchariferous corn media subjected to alcoholic fermentation byMucor X-I culture and in aged sweet wines by means of GLC., GLC-MS, and TLC., as well as by UV and IR spectroscopic examinations. These substances are products of enzymatic esterification of sugars followed by their dehydration by heat treatment or long seasoning of grape wines.
Conversion of lignins from solid parts of grapes during alcoholic fermentation by M. G. Bezhuashvili; R. K. Nutsubidze; M. S. Pataraya (pp. 142-145).
Conversion of lignins contained in solid parts of Rkatsiteli grapes (crests, seeds, and skin) during alcoholic fermentation by wine yeast in Reader’s medium was studied. Various species of wine yeast were used:Saccharomyces oviformis, S. vini Kakhuri 42,S. chodati Teliani 79, andS. uvarum Tsinandali 77. We found that lignins from solid parts of grapes are partially decomposed during alcoholic fermentation, which releases low-molecular-weight aromatic compounds into the medium. A peculiar feature of lignin decomposition during alcoholic fermentation is the formation of reduction products.
Extracellular proteinase and chitinase produced by a culture ofStreptomyces kurssanovii by A. V. Il’ina; N. Yu. Tatarinova; V. E. Tikhonov; V. P. Varlamov (pp. 146-149).
Extracellular enzymes—a chitinase and a proteinase with molecular weights 22 and 32 kDa, respectively—were isolated fromStreptomyces kurssanovii cells. After purification on modified regenerated chitin, the enzymes were virtually homogeneous according to denaturing PAGE. Both enzymes were found to degrade chitosan.
Microbial degradation of components of sewage from phenol production facilities by I. N. Singirtsev; E. V. Volchenko; V. I. Korzhenevich; A. P. Gumenyuk; A. Yu. Fedorov (pp. 150-159).
Processes of aerobic biodegradation of components of phenol production sewage (phenol, acetophenone, dimethylphenylcarbinol, cumene hydroperoxide, α-methylstyrene, benzoate, andp-hydroxybenzoate) by bacterial strains obtained from the collection of the Saratov Institute of Biocatalysis were studied. The metabolic reactions were shown to be oxidative and to have a common catabolic sequence (cumene hydroperoxide-dimethylphenylcarbinol-α-methylstyrene-acetophenone-phenyl acetate-phenol-pyrocatechol-aromatic ring breakage). Benzoate andp-hydroxybenzoate were degraded through the formation of pyrocatechol and protocatechuate, respectively. Metabolic pathways were similar in model mixtures of components and sewage samples.
Study of microbial degradation of nonionic surfactants in designing sewage purification technologies by L. V. Panchenko; O. V. Turkovskaya (pp. 160-164).
Studies of degradation of nonionic surfactants (NISA) in a model purification plant of an original design demonstrated a high rate and depth of degradation processes compared with periodic cultivation of free or immobilized degrading strains. Virtually complete primary degradation (99–99.5%) with destruction of the oxyethyl moiety of the molecule was observed. In addition, NISA molecules were degraded to a greater extent, including considerable degradation of the hydrocarbon radical, partial degradation of aromatic structures in Neonol, and utilization of biologically “unyielding” fractions of commercial NISA preparations, i.e., polyethylene glycol (PEG) and long-chain fractions of polymer homologues.
Effects of composition of the gaseous phase on the formation of hydrocarbons inDesulfovibrio desulfuricans by T. V. Bagaeva (pp. 165-168).
Changes in the synthesis of extracellular metabolic products generated by sulfate-reducing bacteriaDesulfovibrio desulfuricans grown on a lactate-containing mineral medium in the presence of H2 and CO2 at various volume ratios in the gaseous phase were studied. An increase in the amount of extracellular products synthesized by the bacteria was observed at an H2/CO2 ratio of 3 : 1. High concentrations of molecular hydrogen (80–95%) in the presence of 5–20% CO2 facilitated the synthesis of hydrocarbons (alkanes) whose highest concentrations were produced at an H2/CO2 ratio of 9:1. An increase in the initial CO2 concentration in the gaseous phase above 20% increased the amount of oxygenated compounds in the culture.
Effects of conditions ofPenicillium funiculosum G-15 cultivation on production of extracellular glucose oxidase by R. V. Mikhailova; A. G. Lobanok; Zh. F. Shishko; M. I. Yasenko (pp. 169-172).
Effects of conditions ofPenicillium funiculosum G-15 cultivation on the production of extracellular glucose oxidase were studied. The data showed that surface and submerged methods of cultivation can be used for obtaining a glucose oxidizing enzyme. The optimum conditions for submerged cultivation (25°C., initial pH 5.0, and aeration of 3 1/1 per min) and surface cultivation (temperature 25°C and initial pH 4.0) providing the maximum levels of glucose oxidase synthesis were determined.
Effects ofPseudomonas rathonis T cultivation conditions on the functional performance of a biosensor for anionic surfactants by L. A. Taranova; I. N. Semenchuk; P. V. Il’yasov; A. N. Reshetilov (pp. 173-176).
The dependence of the sensitivity of a microbial biosensor of anionic surfactants (AS) on the growth phase ofPseudomonas rathonis T, a strain capable of degrading surfactants, was studied. Correlations were found between the optimum values of temperature and pH for microbial growth, substrate utilization, and functional performance of the microbial biosensor. These results allow the process of AS detection to be optimized.
Ochratoxin A: Contamination of grain by G. P. Kononenko; A. A. Burkin; E. V. Zotova; N. A. Soboleva (pp. 177-180).
Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.
Effect of methyl jasmonate on arachidonic acid-induced resistance of potato to late blight by L. I. Il’inskaya; G. I. Chalenko; E. A. Perekhod; N. G. Gerasimova; O. L. Ozeretskovskaya (pp. 181-186).
Methyl ester of jasmonic acid (Me-JA) influences the induced resistance of potato tubers to late blight caused byPhytophthora infestans. Treatment of potato tuber disk surfaces with Me-JA solution or exposure to an atmosphere containing Me-JA vapors (10−6–10−5 M) increased the rate of rishitin biosynthesis induced by arachidonic acid orP. infestans. Methyl jasmonate increased the sensitivity of potato tissue to arachidonic acid. As a result, in the presence of Me-JA, the protective properties of arachidonic acid were observed at lower concentrations than in the absence of Me-JA. In addition, Me-JA reduced the adverse effects of lipoxygenase inhibitors (salicylhydroxamic acid and esculetin) on the induced resistance of potato tubers to late blight. Therefore, the synergistic interaction of Me-JA and biogenic elicitors can be regarded as part of a mechanism of potato defense against diseases.
Production and immunochemical characterization of monoclonal antibodies toCorynebacterium sepedonicum, the causative agent of potato ring rot by S. M. Ambrosova; V. V. Pavlova (pp. 187-190).
Five stable hybridoma lines producing monoclonal antibodies toCorynebacterium sepedonicum were obtained. The specificity of monoclonal antibodies obtained was characterized. Interactions of the antibodies with native cells and antigenic preparations from bacterial cell extracts were studied. The epitope specificity of these antibodies to their recognized antigens and the use of the antibodies in advanced immunodiagnostic assays are discussed.
Activity of a protein inhibitor of polygalacturonase in potato plants by E. M. Glinka; M. A. Protsenko (pp. 191-193).
The activity of a protein inhibitor of polygalacturonase (PIPG) was studied in potato tubers during storage and in potato leaves and stems during vegetation. The activity of PIPG in tubers varied between seasons. The activity of PIPG during dormancy changed depending on the storage stage and temperature. As a rule, it was higher in etiolated sprouts than in the tubers. The activity of PIPG was slightly higher in leaves of adult vegetating plants than in stems and decreased by the end of vegetation. These changes in the activity of PIPG are suggested to be associated with changes in the growth rate.
Effects of Exin on the development of microbial infection and ethylene release in plants by Hua Kvuet Chian; Nguen Tien Thang; Ngo Ke Syong; E. A. Bulantseva; M. A. Protsenko; E. G. Sal’kova (pp. 194-196).
Effects of Exin on infection of tomato, potato, and cabbage plants withPseudomonas solanacearum andErwinia carotovora and a fungusSclervtium rolfsii were studied. The treatment of infected plants with Exin caused no significant effect on the development of the disease. Treatment with streptomycin as a standard for comparison completely inhibited the growth of these microorganisms. Pretreatment with Exin for one to eight days before infection inhibited the development of diseases. The numbers of tomato and potato plants damaged among those infected withP. solanacearum were lower by 10 and 35% respectively. In field experiments (350 plants per variant), treatment with Exin decreased the development of wilt caused byS. rolfsii andP. solanacearum and rot caused byE. carotovora. Treatment with Exin activated the release of ethylene for not less than 30 days. Possible mechanisms of the effects of Exin are discussed.
Effects of exogenous quercetin on the levels of carbohydrates and amino acids in fruits ofLycopersicon esculentum by L. F. Stakhov; L. N. Stakhova; V. G. Ladygin (pp. 197-200).
Low doses of the exogenous flavonoid quercetin increased the content of sugars in tomato fruits of various cultivars. The content of glucose in tomato fruits of cv.Ukrainskii teplichnyi increased from 3.62 to 11.24% per unit dry weight. Increases in the content of glucose were found in all tomato cultivars examined. An analysis of qualitative and quantitative compositions of amino acids showed that their levels were markedly decreased in fruits of quercetin-treated plants. Our studies and data found in the literature suggest that this effect is due to the synthesis of sugars from amino acids by gluconeogenesis. The reverse process of sugar hydrolysis does not occur because exogenous quercetin inhibits the activity of pyruvate kinase.
Production of agar and agarose from the red algaAhnfeltia tobuchiensis by S. V. Sukhoverkhov; I. A. Kadnikova; A. V. Podkorytova (pp. 201-203).
A simple and inexpensive procedure of agar and agarose production from the red algaAhnfeltia tobuchiensis was developed, which needs no additional Chromatographic purification.