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Applied Biochemistry and Microbiology (v.38, #4)
Dedicated to the memory of I.V. Berezin and R.V. Feniksova Microbial Cellulases (Review) by M. L. Rabinovich; M. S. Melnik; A. V. Bolobova (pp. 305-322).
Compositions of cellulase-hemicellulase systems of aerobic fungi (hyphomycetes, ascomycetes, and basidiomycetes), aerobic bacteria, actinomycetes, as well as anaerobic fungi and bacteria, are considered in the context of the modern structural classification of glycosyl hydrolases. A new nomenclature of cellulases and relative enzymes based on their structural classification is reviewed. Some opportunities of cellulase improvement by means of protein engineering are discussed.
Kinetic Characterization of Extracellular Catalases fromPenicillium piceum F-648 and Its Hydrogen Peroxide-Adapted Variants by A. N. Eremin; D. I. Metelitsa; I. V. Moroz; Zh. I. Pavlovskaya; R. V. Mikhailova (pp. 322-327).
A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (V max), V max/K M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k in, s–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k in *, s–1) was determined at 45°C. In all samples, the specific activity and K M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10–4 mg/ml. The effective constants describing the rate of H2O2 degradation (k, s–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.
Kinetic Characterization of Enzymatic Hydrolysis of Complex Protein Substrates for Producing Nutrient Media by A. D. Neklyudov; A. V. Berdutina; A. N. Ivankin; B. S. Karpo (pp. 328-334).
Hydrolysis of a protein mixture from muscle and bone tissues with the enzymatic system from porcine pancreatic cell suspension was studied. Kinetic constants and the values of activation energy were determined for individual processes of the release of 15 amino acids. The kinetic characteristics of the overall enzymatic hydrolysis calculated from analysis of the changes in concentrations of terminal amino groups were compared with the characteristics obtained while studying the accumulation patterns of individual amino acids.
Isolation of a Kluyveromyces lactis Mutant Enriched in S-Adenosyl-L-Methionine and Growing in Whey Medium by K. P. Mincheva; V. M. Balutsov (pp. 335-338).
The ability of six lactose-digesting yeast strains to accumulate S-adenosyl-L-methionine (AdoMet) when grown on whey medium was studied.Kluyveromyces lactis ATCC 8585 accumulated the greatest amount of AdoMet (15.2 mg per gram biomass) and was chosen for the development of an Ado-Met-enriched mutant. The Ado-Met-enriched mutant AM-65 was selected from mutants induced with N-methyl-N-nitro-N"-nitrosoguanidine. The strain accumulated 61.6 mg AdoMet per gram biomass. The effect of concentrations of medium components on AdoMet biosynthesis was studied. The ability of the mutant to accumulate AdoMet remained stable after multiple reinoculations.
Use of Methylotrophic Bacteria Methylobacillus flagellatumKT for Isolation of Deuterated Exogenous Carbohydrates by I. M. Mishina; A. B. Pshenichnikova; V. I. Shvets; D. A. Skladnev; Yu. D. Tsygankov (pp. 339-346).
Cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum KT were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by HPLC and NMR spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, whereas deuterated exogenous polysaccharides contained 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.
Maleic Acid Utilization by Mixed Cultures of Microorganisms by I. Yu. Safronova; E. V. Semenova (pp. 346-348).
In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and B. subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions of water.
Lipogenesis in the Basidiomycetes Pleurotus ostreatusand Flammulina velutipes Cultivated on Different Media by L. A. Bespalova; O. E. Makarov; L. P. Antonyuk; V. V. Ignatov (pp. 349-354).
The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced byP. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatuswas dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipesshowed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.
Enzyme Immunoassay of Herbicide Decomposition by Soil and Wood Decay Fungi by O. V. Koroleva; E. V. Stepanova; E. O. Landesman; L. G. Vasilchenko; V. V. Khromonygina; A. V. Zherdev; M. L. Rabinovich (pp. 355-360).
The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicilliumsp. 13 and noncellulolytic ones Humicola lanuginosaspp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using the enzyme immune assay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. HyphomyceteMycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of the thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80–92% in 40 days. Mycelia steriliaINBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.
Effect of Ultrasound Treatment on the Increase in the Yield of Amino Acid Mixtures from Ethanol-Assimilating Yeasts by N. I. Belousova; V. S. Orlova; S. V. Gordienko (pp. 361-367).
We studied the effects of ultrasound treatment on an increase of the yield of amino acid mixtures from autolysates of Saccharomyces cerevisiae VKM-Y-2465 grown on ethanol. Adding 2% chloroform (activating agent), autolysis for 5–10 h, and sonication of autolysates allowed us to obtain mixtures balanced in lysine, methionine, and tryptophan and containing up to 90% free amino acids.
Formation and Function of the Legume–Rhizobium Symbiosis of Soybean Plants while Introducing Bacterial Strains from the Genera Azotobacter and Bacillus by N. N. Mel'nikova; L. V. Bulavenko; I. K. Kurdish; L. V. Titova; S. Ya. Kots' (pp. 368-372).
The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicumstrains on the formation and function of the legume–rhizobium symbiosis of soybean plants were studied. The data showed that the bacterial compositions B. japonicum 634b +B. subtilis 5, B. japonicum 634b + A. chroococcum 20, and B. japonicum 10k + A. vinelandii56 with a cell ratio of 1 : 0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all cases by 22–105% compared with the control variants. These binary microbial cultures may be used for the development of combined bacterial preparations for soybean.
Protective Role of Kartolin-4 in Wheat Plants Exposed to Soil Draught by O. F. Monakhova; I. I. Chernyad'ev (pp. 373-380).
Parameters of photosynthesis, heat-resistance, and osmotic pressure of cell exudate of leaves of the drought-sensitive cultivarLyutestsens 758 of wheat, Triticum aestivum, were studied under conditions of normal water supply, soil drought, and subsequent rehydration. The plants preliminarily treated with kartolin-4 were compared to untreated plants. Kartolin-4, a preparation with cytokinin-like activity, partially prevented the drought-induced inhibition of the photosynthetic assimilation of carbon dioxide and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), the key enzyme of carbon metabolism in plants. Upon subsequent rehydration, kartolin-4 stimulated the reparation reactions and facilitated the rapid recovery of normal photosynthetic activity. Kartolin-4 also increased the plant resistance to overheating and water deficiency.
Maize Response to Salicylic Acid and Fusarium moniliforme by O. O. Molodchenkova; V. G. Adamovskaya; Yu. A. Levitskii; O. V. Gontarenko; V. M. Sokolov (pp. 381-385).
Effects of salicylic acid and Fusarium moniliformeon trypsin inhibitor activity, lectin activity, lectin carbohydrate specificity, and salicylic acid content in maize seedlings were studied. Changes in trypsin inhibitor activity, lectin activity, and the content of endogenous salicylic acid after administration of exogenous salicylic acid or a pathogen were shown to depend on the resistance of maize strains to Fusarium. The data suggest that salicylic acid is involved in the induction of trypsin and lectin inhibitors that are important in the formation of defenses against abiotic and biotic factors in maize sprouts.
Wheat Germ Lectin Sorption by Chitosan Polyelectrolyte Complexes by N. A. Samoilova; M. A. Krayukhina; I. A. Yamskov (pp. 386-389).
The possible application of polyelectrolyte complexes (PEC) of chitosan and copolymers of maleic acid with N-vinylpyrrolidone, styrene, and ethylene and/or cross-linked chitin sorbents (CLCS) synthesized on the basis of PEC for sorption of wheat germ lectin (WGL) was studied. The synthesis of spherically granulated sorbents was shown. Compared to unmodified chitosan, there was a significant increase in sorption capacity of WGL by the sorbents: PEC, 2.5-fold, and CLCS, from 3.5-fold to 7-fold.
The Use of Cellocandin, an Enzymatic Preparation from Geotrichum candidum 3C-106, for Defibring Waste Paper and Desiccation of Cellulose Suspension by V. V. Lapin; N. A. Rodionova; N. A. Zagustina; A. T. Kapanchan; N. V. Dubovaya; A. M. Bezborodov (pp. 390-391).
The effect of cellocandin, an enzymatic preparation from Geotrichum candidum 3C-106 with cellulolytic and xylanolytic activities, on defibring of waste paper and acceleration of desiccation of paper pulp was studied. It was shown that cellocandin accelerated defibring of waste paper to cellulose fibers and desiccation of cellulose suspension.
Modified Method for the Determination of Grain Amylase Activity by the Falling Number by I. S. Vorob'eva; M. P. Popov (pp. 392-394).
The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate–amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.
A Photometric Assay for Ethanol by Yu. V. Rodionov; O. I. Keppen; M. V. Sukhacheva (pp. 395-396).
A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.