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Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K m) and maximum reaction velocities (V max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K i = 104.7 ± 21.7 mM, K m = 2.5 ± 0.5 mM, V max = 25.1 ± 1.5 U/mg) was observed.

The amidase of Rhodococcus rhodochrous 4-1 was immobilized by covalent attachment to activated chitosan by physical sorption on carbon adsorbents and by the formation of crosslinked aggregates in the absence of carrier material. Comparative analysis of particular catalytic properties of the free and chitosan-immobilized amidase was performed. It was shown that the enzyme retained 50-60% of its initial activity after covalent immobilization on chitosan and was characterized by increased temperature stability as compared to soluble amidase. Moreover, the immobilized enzyme retained more than 20% of its activity after five 24-h cycles of acrylamide deamination. The effects of different types of immobilization on amidase stereose-lective properties were studied by the model reaction of racemic lactamide hydrolysis to D-lactic acid and L-lactic acid. It was shown that crosslinked amidase aggregates possessed high D-stereoselectivity (up to 77-94%). The immobilized enzyme showed the highest enantioselectivity at 60 degrees C.

We report the development of a heterogeneous biocatalyst for the hydrolysis of amides that is based on cell adhesion ofamidase-containing Rhodococci on activated birch carbon (ABC) and crude carbon. We investigated the properties of the obtained biocatalyst in the hydrolysis reaction of acrylamide to acrylic acid and nicotinamide to nicotinic acid, as well as in a model reaction of racemic lactamide hydrolysis to a mixture of D- and L-isomers of lactic acid. We show that a six- and threefold increase in the concentrations of adherent and suspended cells, respectively, results in a reduction of amidase activity by 3 and 30 times, respectively. ABC Cells adherent on ABC maintained more than 50% of enzymatic activity for seven 24-hour cycles of acrylamide hydrolysis, while suspended cells lost more than 60% of activity already in the second cycle. We also noted that cell adhesion on ABC reduced the stereoselectivity of hydrolysis reaction of racemic lactamide.

A new aliphatic amidase gene (ami), having a level of similarity with the nearest homologs of no more than 77%, was identified in the Rhodococcus erythropolis TA37 strain, which is able to hydrolyze a wide range of amides. The amidase gene was cloned within a 3.7 kb chromosomal locus, which also contains putative acetyl-CoA ligase and ABC-type transportergenes. The structure of this locus in the R. erythropolis TA37 strain differs from the structure of loci in other Rhodococcus strains. The amidase gene is expressed in Escherichia coli cells. It was demonstrated that amidase (generated in the recombinant strain) efficiently hydrolyzes acetamide (aliphatic anmide) and does not use 4'-nitroacetanilide (N-substituted amide) as a substrate. Insertional inactivation of the amidase gene in the R. erythropolis TA37 strain results in a considerable decrease (by at least 6-7 times) in basal amidase activity, indicating functional amidase activity in the R. erythropolis TA37 strain.

The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.

The degradation of functionalized arenes exposed to a mixed culture of chemoorganoaeroheterotrophic microorganisms has been shown to predominantly occur through oxidative intradiol decyclization of the aromatic ring. The spatial structure of the catalytic center of intradiol dioxygenase and its complexes with catechol and protocatechuic acid has been characterized using quantum chemical modeling procedures. The dependence of the total energy of the enzyme-substrate complex on the length of the bond between the iron atom and the hydroxyl group of the substrate (Fe-4-OH and Fe-1-OH) before and after the separation of water and a tyrosine residue (Tyr) from the catalytic center has been demonstrated for the first time.

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

Lipolytic and catalase activity of Pseudomonas pseudoalcaligenes 109, Rhodococcus erythropolis 102, Bacillus subtilis 138 and their association with different growth models: biofilm and plankton ones. It is shown that under biofilm conditions the fermentative activity of bacteria under study was 1.5-1.7 times higher than under plankton conditions. Monocultures of bacteria displayed much lower activity than associative ones. Changes of physico-chemical properties of the specimens of protective coating Polyken 980-25 with participation of the above bacteria have been studied. The coating breaking strength decreases by 5.9-11.8% under the effect of monocultures, and by 17.3% under the effect of association. The adhesive strength as the basic index of coating biologic resistance decreased, respectively, in mono- and associated cultures by 28.6-73.2% in respect of the control. Damaging the sticking layer of isolation coating, bacteria damage the adhesion to metal which favors its corrosion.

The dynamics of catalase activity of the hydrocarbon-oxidizing bacteria Cordona terrae, Rhodococcus rubropertinctus, and Rhodococcus erythropolis during petroleum product destruction has been studied. A direct relationship between decreasing catalase activity of hydrocarbon-oxidizing microorganisms and the intensity of petroleum product destruction has been established experimentally. The revealed dependence allows one to consider the catalase activity of bacteria as an indicator of the initial stage of petroleum product oxidation and may be used for choosing destructor strains to construct biopreparations suitable for natural ecosystem remediation.

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters ofacrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (Vmax), whereas the Michaelis constant (K(M)) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0-4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.

It was established that glycerol, a byproduct of biodiesel production, may be used as substrate for synthesis of surfactants Rhodococcus erythropolis IMV Ac-5017 and Acinetobacter calcoaceticus IMV B-7241. Maximum indices of surfactants synthesis by the strain IMV B-7241 have been fixed, when the medium with glycerol included yeast autolysate and trace elements. It was shown that the surfactants synthesis could be intensified when cultivating A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 on the mixture of hexadecane and glycerol in concentration of 0.5-1.0% (in volume). When using inoculate grown on hexadecane, the conditional concentration of the surfactant A.calcoaceticus IMV B-7241 on the mixed substrate was higher by 56-100, and that of R. erythropolis IMVAc-5017 by 260-320 % than on the monosubstrate glycerol. The paper is presented in Russian.

The nitrile hydratase isolated from Rhodococcus ruber strain gt1, displaying a high nitrile hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon carrier Sibunit. The activity and operational stability of the immobilized nitrile hydratase were studied in the reaction of acrylonitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the carrier led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The nitrile hydratase immobilized on Sibunit and carbon-containing aluminum alpha-oxide having a "crust" structure displayed the highest operational stability in acrylonitrile hydration. It was shown that the thermostability of adsorbed nitrile hydratase increased by one order of magnitude.

A 9alpha-hydrolase activity of a new actinobacterium strain identified as Rhodococcus erythropolis based on the analysis of a 16S rRNA gene sequence (1417 nucleotides) towards androst-4-en-3,17-dione (AD) was studied. In the presence of glucose in the medium, this strain completely transformed AD (4-20 g/l) into 9alpha-hydroxy-AD over 20-48 h. This culture was able to grow and perform AD 9alphahydroxylation at a concentration of dimethyl formamide up to 9%. Crystalline 9alpha-hydroxy-AD was isolated with a yield of over 90%.

During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two pyrocatechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to pyrocatechol 1,2-dioxygenases of the classical ortho-pathway. Chloropyrocatechols and chlorophenols served as competitive antagonists of pyrocatechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.

Four types of alcohol- and acetaldehyde dehydrogenases were found in the cells of strain Rhodococcus erythropolis EK1 grown on ethanol. They are as follows: NAD-, NADP-, pyroquinoline quinone (PQQ)- and 4-nitroso-N,N-dimethyl aniline (NDMA)-dependent enzymes. Activity of NAD- and NADP+ -dependent alcohol dehydrogenases, as well as PQQ and NDMA-dependent acetaldehyde dehydrogenases was low and made up 3-11 nmol x min(-1) x mg(-1) of protein. Ethanol oxidation in the given strain is realized by NDMA-dependent alcohol dehydrogenase, which activity reached its maximum value (up to 15 nmol x min(-1) x mg(-1) of protein) in the early exponential growth phase. Acetaldehyde is oxidized with participation of NAD and NADP-dependent dehydrogenases with optimum pH 9.0-9.5. The results obtained are a basis for development of approaches to intensification of surfactants synthesis by Rhodococcus erythropolis EK-1 strain.

Screening of alkane hydroxylase genes (alkB) was performed in the thermophilic aerobic bacteria of the genus Geobacillus. Total DNA was extracted from the biomass of 11 strains grown on the mixture of saturated C10-C20 hydrocarbons, PCR amplification of fragments of alkB genes was performed with degenerate oligonucleotide primers, PCR products were cloned and sequenced. For the first time in the genome of thermophilic bacteria the presence of a set of alkB gene homologues was revealed. The strains each contain three to six homologues among which only two are universal for all of the strains. Comparative phylogenetic analysis of the nucleotide sequences and the inferred amino acid sequences showed close relatedness of six of the revealed variants of geobacilli sequences to the alkB4, alkB3, and alkB2 genes that had previously been revealed by other authors in Rhodococcus erythropolis strains NRRL B-16531 and Q15. The rest two variants of alkB sequences were unique. Analysis of the GC composition of all the Geobacillus alkB homologues revealed closer proximity to the rhodococcal chromosomal DNA than to the chromosomal DNA of geobacilli. This may be an indication of the introduction of the alkB genes into the Geobacillus genome by interspecies horizontal transfer; and rhodococci or other representatives of the Actinobacteria phylum were probably the donors of these genes. Analysis of the codon usage in fragments of alkB genes confirms the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. Formation of a set of several alkB homologues in a genome of a particular microorganism may result from free gene exchange within this pool.

Rhodococcus ruber strain gtl, possessing nitrile hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the nitrile hydratase reaction (acrylonitrile), and the product (acrylamide) was studied. Also, the effect of immobilization and nitrile hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents.

The amidase gene from Rhodococcus rhodochrous M18 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.

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 beta-sitosterol (BSS) and its 3beta-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 17beta-hydroxyandrost-4-ene-3-one (testosterone), commonly used in pharmacology, were determined.

The bacteriological study of specimens taken from environmental objects, biomaterials from animals and sputa from tuberculosis patients revealed the ubiquity of Mycobacterium, Nocardia and rhodococci obtained from different kinds of environment in the Republic of Daghestan. In the identification 83.6% out of the isolated atypical Mycobacterium cultures were classified with Ranion groups II and IV. Among Nocardia, the most widespread organisms were N. asteroides (62.7%) and among rhodococci, R. erithropolis (61.9%).

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase isolated from the Rhodococcus sp. strain gt1.

The ability of propane-assimilating microorganisms of the genus Rhodococcus to utilize metabolites of the terminal and subterminal pathways of propane oxidation was studied. Propane monooxygenase of Rhodococcus erythropolis 3/89 was shown to be the an inducible enzyme catalyzing epoxidation and hydroxylation of organic compounds. The optimum conditions for epoxidation of gaseous and liquid alkenes and hydroxylation of aromatic carbohydrates were found.

The ability of propane monooxygenase of Rhodococcus erythropolis 3/89 to catalyze oxidation of higher liquid alkenes and aromatic hydrocarbons was studied. Optimal conditions of tetradecene epoxidation and benzene hydroxylation were found. Under these conditions, oxidation was shown to be accompanied by a 100% conversion of benzene to phenol.

Nitrile hydratase was isolated and purified to homogeneity from cells of Rhodococcus rhodochrous M8. This enzyme catalyzes the hydrolysis of acrylic acid nitrile to acrylamide. Nitrile hydratase content in the cell was shown to be 17% of total soluble protein. The molecular weight of the native enzyme was 510 kDa. The enzyme consisted of two subunits with molecular weights of 23.5 kDa and 28.0 kDa. The N-terminal amino acid sequences of these subunits were estimated.

Active sulphate-reducing microorganism which belongs to the genus Pseudomonas has been distinguished and described. The culture is a facultative aerobe, optimum Eh is -170-180 mV. Pseudomonas sp. being cultivated under strictly anaerobic conditions sulphate-reduction proceeds more intensively than under aerobic conditions. This fact should be taken into account under treatment of industrial sewage.

Blast transformation of peripheral blood lymphocytes in healthy people is studied by enzymes of the microbic origin possessing the lectin activity. Galactose oxidase of Fusarium graminearum IMV-F-1060 is shown to be mitogenically active with respect to the lymphocytes in the culture in vitro and may be one of home sources of lymphocytic mitogens for the laboratory investigations.

The regulation of terephthalate catabolism was studied in Rhodococcus rubropertinctus which decomposed this synthetic monomer. The pathway (a) of terephthalate (TP) catabolism is as follows: TP----benzoate----4-hydroxybenzoate----protocatechuate----pyrocatechol-- --cycle ortho-cleavage. The following results were obtained when studying why two other catabolic pathways were realized if benzoate and 4-hydroxybenzoate were taken as a sole carbon source, namely, (b) benzoate----pyrocatechol----cycle cleavage and (c) 4-hydroxybenzoate----protocatechuate----cycle cleavage. TP seemed to cause the divergence of pathways (a) and (b) by repressing the system of benzoate oxidation to pyrocatechol. In pathway (c), benzoate repressed the synthesis of enzymes which catalysed protocatechuate oxidation. Pathway (b) was switched over to (a) when the strain was grown in a medium containing TP and benzoate at a benzoate concentration above 5 mM. Here, the concentration of benzoate (first exogenous and later formed from TP) played a key role. R. rubropertinctus growth in a medium with TP and glucose had diauxic characteristics.

The activity of esterase was studied in bacteria oxidizing hydrocarbons and belonging to the genera Rhodococcus, Arthrobacter and Pseudomonas. Indophenyl acetate was used as a substrate of the reaction catalysed by the enzyme. Exocellular esterases were not found. Endocellular esterases differed in their activity and thermostability both among the genera and among species of one and the same genus.