[Morphological, Physiological, and Biochemical Characteristics of a Benzoate-Degrading Strain Rhodococcus opacus 1CP under Stress Conditions].

Ability of actinobacteria Rhodococcus opacus 1CP to survive under unfavorable conditions and retain its biodegradation activity was assessed. The morphological and ultrastructural features of R. opacus 1CP cells degrading benzoate in the presence of oxidants and stress-protecting agents were investigated. The cells of R. opacus 1CP were resistant to oxidative stress caused by up to 100 mM H2O2 or up to 25 µM juglone (5-oxy-1,4-naphthoquinone). After 2 h of stress impact, changes in the fatty acid composition, increased activity of antioxidant enzymes, and changes in cell morphology and ultrastructure were observed. The strain retained its ability to degrade benzoate. Quercetin had a protective effect on benzoate-degrading cells of R. opacus 1CP. The strategy for cells survival under unfavorable conditions was formulated, which included decreased cell size/volume and formation of densely-packed cell conglomerates, in which the cells are embedded into a common matrix. Formation of conglomerates may probably be considered as a means for protecting the cells against aggressive environmental factors. The multicellular conglomerate structure and the matrix material impede the penetration of toxic substances into the conglomerates, promoting survival of the cells located inside.

[Xylanase and cellulase of fungus Cerrena unicolor VKM F-3196: production, properties, and applications for the saccharification of plant material].

Under the conditions of submerged cultivation in a medium containing microcrystalline cellulose, the Cerrena unicolor VKM F-3196 basidiomycete is capable of producing xylanase and cellulase. Electrophoretically homogeneous cellulase and xylanase were obtained using ion exchange and hydrophobic chromatography. The molecular weight of both cellulase and xylanase was -44 kDa. It was shown that xylanase catalyzed the hydrolysis of xylan with the production of xylose, xylobiose, and xylotetrose and it exhibited properties of endoxylanases. Cellulase hydrolyzed carboxymethylcellulose, xylan, and microcrystalline cellulose with the formation of cellotriose and cellotetraose. For both enzymes, the pH optimum was -4.0. The enzymes exhibited moderate thermostability: xylanase retained 35% of the initial activity for an hour at 60 degrees C; cellulase, 10% under the same conditions. Xylanase, cellulose, and a mixture of these enzymes saccharified plant material (wheat, rye, wheat middling, and oat), indicating the possible use of these enzymes in biotechnology.

[Immobilization of a recombinant strain producing glucose isomerase on SiO2-xerogel and properties of prepared biocatalysts].

An original method of immobilization of nongrowing microorganism cells on xerogel of silicon dioxide containing insoluble hydroxyl compounds of cobalt(III) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3-5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62-65 degrees C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was -60 and -25 days, respectively.

[Xylose isomerase constitutive biosynthesis in Arthrobacter nicotianae].

The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product ofxylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.

[Catalytical properties of Arthrobacter nicotianae cells, a producer of glucose isomerase, immobilized in xerogel of silicium dioxide].

Arthrobacter nicotinanae cells, producers of glucose isomerase, were immobilized in xerogel of silicium dioxide, and properties of the resulted heterogeneous biocatalysts were investigated in the process of isomerization of monosaccharide (glucose and fructose). The glucose isomerase activity of the resulted biocatalysts was shown to be 10 U/g, on average, taking into account the loss of the activity upon the immobilization, which amounted to 50% of the cell activity in suspension. The rate of the fructose isomerization increased linearly in the range of 55-80 degrees C with the temperature coefficient 1.3. The biocatalysts were stable in this range; they were rapidly inactivated, however, at increasing temperature. The half-inactivation time was six to seven h and five min or less at 80 degrees C and 85 degrees C, respectively. The half-inactivation time of heterogeneous biocatalysts was 50-90 h in the periodic process of isomerization of 2 M monosaccharides at 60 degrees C in the presence of the immobilized Arthrobacter nicotinanae cells.

[Glucose isomerase activity in suspension of Arthrobacter nicotianae cells and adsorption immobilization of the microorganisms on inorganic carriers].

Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 micromol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60-80 degrees C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foam ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70 degrees C. Maximum glucose isomerase activity (2 micromol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.

[Catabolite repression of xylose isomerase synthesis in Arthrobacter ureafaciens].

The effect of a specific substrate as well as other carbon sources on the biosynthesis of xylose isomerase in the actinobacterium Arthrobacter ureafaciens BIM B-6 has been studied. It was established that xylose and its structural analogue xylite induced the production of the enzyme by bacterial cells. The inducing effect peaked at a concentration of specific substrates of 0.025% (as carbon) and then remained unchanged irrespective of the substrate amount. It has been shown that the synthesis of xylose isomerase by A. ureafaciens is controlled by catabolite repression occurring at the transcription level and mediated by cyclic 3',5'-AMP.

[Spontaneous variability of glucose oxidase-producing fungus Penicillium adametzii LF F-2044].

The glucose oxidase-producing fungus Penicillium adametzii LF F-2044 was studied for natural variability. Four variants of the fungus differed in morphological characteristics and glucose oxidase synthesis. The synthesis of extracellular glucose oxidase and the productivity of morphological variants P. adametzii LF F-2044.1 and P. adametzii LF F-2044.2 were 127-146 and 95-159% higher, respectively, than the control. Highly active morphological variants of the fungus were chosen for further selection experiments.

[Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovota subsp. atroseptica].

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.

[Growth characteristics and glucose oxidase production by mutant Penicillium funiculosum strains]. / Izuchenie zakonomernostei rosta i obrazovaniia glukozooksidazy u mutantnykh shtammov Penicillium funiculosum.

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.

[A plate method to screen for microorganisms producing xylose isomerase]. / Chashechnyi metod skrininga mikroorganizmov--produtsentov ksilozoizomerazy.

A plate method was developed to screen for xylose isomerase-producing microorganisms based on the use of 2,3,5-triphenyltetrazolium as an indicator of D-xylulose, the D-xylose isomerization product. The use of this method allows microorganisms to be differentiated by the character of the enzyme synthesis (inducible or constitutive).

[Xylose(glucose) isomerase reactivity of immobilized Arthrobacter sp]. / Svoistva preparata immobilizovannykh kletok Arthrobacter sp.--produtsenta ksilozo(gliukozo)izomerazy.

The study of the xylose/glucose isomerase-containing Arthrobacter sp. B-5 cells immobilized in cobalt hydroxide gel showed that immobilization increases the substrate affinity of the enzyme, its thermo- and pH-optima of action and stability, and makes unnecessary the addition of stabilizing cobalt ions to the isomerization medium.

[Composition and biological activity of submerged mycelium of the xylotrophic basidiomycete Lentinus edodes]. / Sostav i biologicheskaia aktivnost' glubinnogo mitseliia ksilotrofnogo bazidiomitseta Lentinus edodes.

The composition of submerged mycelium of Lentinus edodes, grown in laboratory fermenters, has been studied. The mycelium contained 23-24% proteins, 8-9% lipids, up to 1800 mg% phenolic substances, and a significant amount of inorganic substances, including calcium and iron. The fungus produced up to 5.0% intracellular and 3.5-4.0 g/l extracellular polysaccharides. The submerged mycelium stimulated the development of humoral immune response elicited by sheep red blood cells.

[Development of the biological preparation enatin with broad-range antimicrobial action]. / Biopreparat énatin s shirokim spektrom antimikrobnogo deistviia.

Physiological and biochemical traits of epiphytic spore forming bacteria Bacillus pumilis BIM V-263 were examined. The nutrient medium and conditions for submerged cultivation of the strain were selected. The growth dynamics and antagonistic activity during cultivation in a laboratory fermenter ANKUM-2M were studied. The results provide grounds for development of the biological preparation Enatin with broad-range antimicrobial effect. The plant-protective and growth-stimulating effect of Enatin was examined in laboratory and field experiments. The preparation holds promise as means for biological control of crop pathogens.

[Selection of Penicillium funiculosum strains with high glucose oxidase activity]. / Otbor shtammov Penicillium funiculosum--vysokoaktivnykh produtsentov gliukosooksidazy.

Metabolic inhibitors, riboflavin, and end products of glucose oxidation were shown to hold much promise for the selection of Penicillium funiculosum mutant strains with a high glucose oxidase activity. The incidence of positive mutations was highest in clones resistant to sodium azide, riboflavin, and beta-D-glucono-delta-lactone. Enzyme activity in Penicillium funiculosum mutants was studied under conditions of submerged cultivation. The intensity of glucose oxidase synthesis in seven cultures was 24-56% higher than that in the parent strain of Penicillium funiculosum NMM95.132.

[Thermal stability of Penicillium adametzii glucose oxidase]. / Termicheskaia stabil'nost' gliukozoksidazy Penicillium adametzii.

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70 degrees C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (< 15 micrograms/ml) and pH > 5.5, the rate constants kin (s-1) for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of beta structures in stabilizing the protein globule. At a concentration of 15 micrograms protein/ml, the activation energy Ea of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60 degrees C had the following values: delta H = 78.4 kcal/mol, delta G = 25.5 kcal/mol, and delta S = 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.

[Preparation and characteristics of mutant strains of Penicillium funiculosum--producers of glucose oxidase]. / Poluchenie i kharakteristika mutantnykh shtammov Penicillium funiculosum--produtsentov gliukozooksidazy.

After the mutagenesis of Penicillium funiculosum with UV light and N-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5-153% higher (in a medium with glucose) and 4-83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutant P. funiculosum NMU95-132 was chosen for further selection work.

[Isoelectric characterization of extracellular polygalacturanases of various Aspergillus alliaceus strains]. / Izoélektricheskaia kharakteristika vnekletochnykh poligalakturonaz razlichnykh shtammov Aspergillus alliaceus.

The composition of pectin hydrolase complexes produced by various Aspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains of A. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.

Effect of aromatic compounds on cellular fatty acid composition of Rhodococcus opacus.

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.

Utilization of Halogenated Benzenes, Phenols, and Benzoates by Rhodococcus opacus GM-14.

Strain GM-14 was isolated by selective enrichment from contaminated soil with chlorobenzene as the sole source of carbon and energy. It utilizes an exceptionally wide spectrum of haloaromatic substrates. It is a gram-positive, weakly acid-fast actinomycete, with a morphological cycle from cocci and short rods to long rods and branched filaments; it grew optimally at 28(deg)C; and it tolerated 5% NaCl in rich medium. The chemotaxonomic characteristics, the diagnostic biochemical tests, the whole-cell fatty acid composition, and 16S rDNA analysis were consistent with Rhodococcus opacus. R. opacus GM-14 grew on 48 of 117 different aromatic and haloaromatic compounds. It utilized phenol at concentrations up to 1.2 g/liter, 3- and 4-methylphenols up to 0.5 g/liter, 2- and 4-chlorophenols up to 0.25 g/liter, and 3-chlorophenol up to 0.1 g/liter. It grew in saturated aqueous solutions of benzene, chlorobenzene, and 1,3- and 1,4-dichlorobenzene (up to 13, 3, 0.5, and 0.5 g/liter, respectively). The specific growth rate of strain GM-14 on phenol and 3- and 4-chlorophenols in batch culture was 0.27 to 0.29 h(sup-1), and that on benzene and chlorobenzene was similar to the rate on fructose, i.e., 0.2 h(sup-1). The growth yield on benzene and on chlorobenzene (<=0.4 g liter(sup-1)) was 40 to 50 g (dry weight) per mol of substrate consumed, equalling 8 g of dry weight biomass per mol of substrate carbon, similar to that obtained on acetate. During growth of strain GM-14 on chlorobenzene, 1,3-dichlorobenzene, and all isomers of monochlorophenol, stoichiometric amounts of chloride were released, and 50% of the stoichiometric amount was released from 1,4-dichlorobenzene.