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M V Donova

Publications and source records attributed to M V Donova.

13 recordsLinked to original sources
PubMed2006

Steroid-1-dehydrogenase of Mycobacterium sp. VKM Ac-1817D strain producing 9alpha-hydroxy-androst-4-ene-3,17-dione from sitosterol.

The strain of Mycobacterium sp. VKM Ac-1817D forms 9alpha-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M (r) approximately 58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.

Ammonium Sulfate↗
PubMed2005

Localization of 17beta-hydroxysteroid dehydrogenase in Mycobacterium sp. VKM Ac-1815D mutant strain.

The localization of mycobacterial 17beta-hydroxysteroid dehydrogenase (17beta-OH SDH) was studied using cell fractionation and cytochemical investigation. Mycobacterium sp. Et1 mutant strain derived from Mycobacterium sp. VKM Ac-1815D and characterized by increased 17beta-OH SDH activity was used as a model organism. Subcellular distribution study showed both soluble and membrane-bound forms of mycobacterial 17beta-hydroxysteroid dehydrogenase. The cytochemical method based on a copper ferrocyanide procedure followed by electron microscopic visualization was applied in order to investigate the intracellular localization of bacterial 17beta-OH SDH in more detail. The enzyme was found to be located in the peripheral cytoplasmic zone adjoining the cytoplasmic membrane (CM). 17beta-OH SDH was loosely membrane bound and easily released into the environment under the cell integrity failure.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

Mycobacterium sp. mutant strain producing 9alpha-hydroxyandrostenedione from sitosterol.

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9alpha-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9alpha-hydroxylase activity.

Androstadienes↗
PubMed2004

Extracellular 3beta-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D.

Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5-->delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

17-Hydroxysteroid dehydrogenases of Mycobacterium sp. VKM Ac-1815D mutant strain.

Whole cells and crude extract of Mycobacterium sp. VKM Ac-1815D mutant strain Et1 were shown to carry out 17beta-reduction, 17beta-dehydrogenation and 1(2)-reduction of 3-keto-C(19)-steroids. Two 17-hydroxy steroid dehydrogenases (17-OH SDH) were partially purified from the strain by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-sephacel and gel-filtration on Bio-Gel A. The enzymes differed in chromatographic properties and specific activities. One enzyme--17-OH SDH (2) (tetramer, M(r) approximately 210,000) was found to be responsible for bi-directional reduction-oxidation of steroids at C 17, whereas the other one--17-OH SDH (1) (monomer, M(r) approximately 68,000) specifically catalysed 17beta-dehydrogenation of 17-hydroxysteroids (testosterone and 1(2)-dehydro testosterone). The 17beta-reduction of 1-ene-17-ketosteroids was accompanied by 1(2)-reduction. A role of 1-ene-reductase as a steroid-binding protein associated with 17-OH SDH (2) in Mycobacterium sp. is discussed.

17-Hydroxysteroid Dehydrogenases↗
PubMed2002

Estimation of cyclodextrin affinity to steroids.

A nonlinear spectrometric method for determination of the stability constant (Ks) for cyclodextrin complex with steroid was developed. The method is based on calculation of the parameters of competitive cyclodextrin complexation by simultaneous fitting of two types of curves. Those of the first type are the dependencies of absorbance of methyl orange solution on the cyclodextrin concentration, the second type being the absorption curves of displacement of the dye, by steroid, from the cyclodextrin complex. With the method proposed, Ks values were calculated with standard deviation less than 10%. This method is validated by determination of Ks values using the phase-solubility technique. For neutral steroid molecules, the effect of pH on Ks was found to be insignificant. Ks values for the cyclodextrin-dye complex were determined for randomly methylated beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin and sulfobutylether-beta-cyclodextrin. More hydrophobic steroids were characterised by higher Ks values. Anionic beta-cyclodextrins showed high affinity for the steroids studied. Simple equipment and sufficient computing allowed recommendation of the method for express estimation of cyclodextrin's affinity for hydrophobic substrates.

Cyclodextrins↗
PubMed2000

Conversion of 1-benzoylindole by Aspergillus strains.

Biotransformation of 1-benzoylindole (BI) by the strains Aspergillus flavus VKM F-1024 and Aspergillus oryzae VKM F-44 was studied. The major metabolites isolated were identified as 4-hydroxyindole (4-HI), 5-hydroxyindole (5-HI), 4-hydroxy- -benzoylindole, 4-hydroxy-1-(4'-hydroxy)-benzoylindole and indole. The structure of the metabolites was determined by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The pathways of BI metabolism via initial monohydroxylation at C-4 and C-5 followed by cleavage of the benzoyl substituent to yield 4-HI and 5-HI were proposed. Indole was formed as a by-product, and its role as a potent inhibitor of BI hydroxylation at C-4 and C-5 is discussed.

Aspergillus↗
PubMed1996

FET-microbial sensor for xylose detection based on Gluconobacter oxydans cells.

A potentiometric biosensor for xylose was devised utilizing Gluconobacter oxydans whole cells. Immobilization methods based on physical adsorption were used for G. oxydans cells and extracellular pH changes resulting from xylose dehydrogenation were monitored by a field effect transistor (FET). The G. oxydans, FET-based sensor detected xylose at a lower limit of 0.5 mM. From 5.0 to 30 mM xylose, the response of the sensor was linear. Expectedly, output signals were significantly suppressed by buffer (Tris-HCl). Responses were essentially stable for at least four weeks of storage and showed only a slight loss of initial xylose sensitivity. Xylitol exerted an insignificant influence on the sensor's response to xylose. However, the response to glucose was 5 times higher in relation to that of xylose at the same concentration (1 mM). For xylose determinations in the presence of glucose, a two-step assay is discussed.

Acetobacteraceae↗
PubMedDate not supplied

[Preparation of fed-batch culture of Nocardioides sp].

A screening for carbon sources revealed ethanol to be the best substrate for condensed Nocardioides sp. culture. A strategy achieving the maximum (21 g/l) yield of biomass was developed for the control over the condensed fed-batch culture production. This control based on the algorithm ExpoDense should be predetermined in the first phase and adaptive in the second phase of two-phase process of condensed culture production.

Algorithms↗
PubMedDate not supplied

[The 1(2)-dehydrogenation of steroid substrates by Nocardioides simplex VKM Ac-2033D].

The bacterium formerly known as Arthrobacter globiformis 193 has high 1(2)-dehydrogenase activity toward pharmaceutically important steroids, 9(11)-dehydrocortexolone in particular. The complex analysis of the morphostructural, physiological, biochemical, and phylogenetic properties of this bacterium allowed us to reclassify it into Nocardioides simplex (N. simplex VKM Ac-2033D).

Arthrobacter↗
PubMedDate not supplied

[Enzymes involved in modification of the steroid nucleus of industrial mycobacterial strains: isolation, functions, and properties].

The key enzymes involved in modification of the steroid nucleus of sterol-transforming mycobacteria--3beta-hydroxysteroid oxidase (3-OH-SO, EC 1.13.1.2) and 17beta-hydroxysteroid dehydrogenase (17-OH-SDH, EC 1.1.1)--were isolated and characterized. It is shown that 3-OH-SO is a multifunctional enzyme catalyzing oxidation of the 3beta-OH group, delta5 --> delta4 isomerization, and 6-hydroxylation. Two forms of intracellular 17-OH-SDH that catalyze redox reactions at C17 were found, and their properties were determined. The presence of an extracellular 17-OH-SDH in Mycobacterium spp. (VKM Ac-1815 D and Et1) was demonstrated for the first time.

3-Hydroxysteroid Dehydrogenases↗