Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, α-chlorophenylacetamide, and α-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Purification and properties of an amidase from Rhodococcus erythropolis MP50 which enantioselectively hydrolyzes 2-arylpropionamides.

An enantioselective amidase from Rhodococcus erythropolis MP50 was purified to homogeneity. The enzyme has a molecular weight of about 480,000 and is composed of identical subunits with molecular weights of about 61,000. The NH2-terminal amino acid sequence was significantly different from previously published sequences of bacterial amidases. The purified amidase hydrolyzed a wide range of aliphatic and aromatic amides, The highest enzyme activities were found with amides carrying hydrophobic residues, such as pentyl or naphthoyl. The purified enzyme converted racemic 2-phenylpropionamide, naproxen amide [2-(6-methoxy-2-naphthyl) propionamide], and ketoprofen amide [2-(3'-benzoylphenyl)propionamide] to the corresponding S-acids with an enantiomeric excess of >99% and an almost 50% conversion of the racemic amides. The enzyme also hydrolyzed different alpha-amino amides but without significant enantioselectivity.     

Nitrile hydrolysis activity of Rhodococcus erythropolis NCIMB 11540 whole cells

The nitrile hydrolyzing properties of the bacterium strain Rhodococcus erythropolis NCIMB 11540 have been investigated. Using whole cells of the microorganism, a wide variety of aromatic and aliphatic cyanide-containing substrates was successfully hydrolyzed to the corresponding amide or acid. In the case of dicyanides, selective monohydrolysis took place, which was further explored in the desymmetrization of malononitriles resulting in the corresponding cyano amides in enantiomeric excesses of up to 98%.     

Cesium Accumulation and Growth Characteristics of Rhodococcus erythropolis CS98 and Rhodococcus sp. Strain CS402

Growth and cesium accumulation characteristics of two cesium-accumulating bacteria isolated from soils were investigated. Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 accumulated high levels of cesium (approximately 690 and 380 μmol/g [dry weight] of cells or 92 and 52 mg/g [dry weight] of cells, respectively) after 24 h of incubation in the presence of 0.5 mM cesium. The optimum pH for cesium uptake by both Rhodococcus strains was 8.5. Rubidium and cesium assumed part of the role of potassium in the growth of both Rhodococcus strains. Potassium and rubidium inhibited cesium accumulation by these Rhodococcus strains. It is likely that both Rhodococcus strains accumulated cesium through a potassium transport system.     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50

The two new bacterial strains, Rhodococcus sp. C3II and Rhodococcus erythropolis MP50, which have been especially selected for the enantioselective hydrolysis of pharmaceutically interesting 2-arylpropionitriles like naproxen nitrile, have been applied for the hydrolysis of various aliphatic and aromatic nitriles and acid amides. From the enantioselective hydrolysis of racemic ibuprofen amide 4, 2-phenylbutyronitrile 5a as well as the profen-related atrolactamide 8 we deduce the decisive role of both an alkyl and aryl substituent in the -position to the nitrile or amide function for high enantioselectivity of the hydrolysis. Strain C3II and MP50 differ in the activity of their nitrile hydratase–amidase enzyme systems. This is of interest for the regioselective hydrolysis of the dinitriles 10a–13a to diacids 10f–13f. While strain C3II is suitable to preferentially produce mononitrile monoamide derivatives, strain MP50 can be used especially to form mononitrile monoacid and monoamide monoacid derivatives.     

Oxidation of organic compounds by Rhodococcus erythropolis 3/89 propanomonooxygenase

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.     

Formation of a Chiral Hydroxamic Acid with an Amidase from Rhodococcus erythropolis MP50 and Subsequent Chemical Lossen Rearrangement to a Chiral Amine

The amidase from Rhodococcus erythropolis MP50 demonstrated, in the presence of hydroxylamine, acyltransferase activity and catalyzed the formation of hydroxamates from amides and hydroxylamine. The rates of acyltransferase activity of the purified amidase for the substrates acetamide, phenylacetamide, and 2-phenylpropionamide were higher than the corresponding rates for the hydrolysis reactions. With the substrate 2-phenylpropionamide the hydrolysis reaction and the acyltransferase activity were highly enantioselective. The optically active 2-phenylpropionhydroxamate was converted by a chemical Lossen rearrangement in an aqueous medium into the enantiopure S-1-phenylethylamine.     

红球菌DS-3脱除二苯并噻吩中有机硫的性能初探

从孤岛油田分离到一株红球菌（Rhodococcus sp.）DS\|3,能专一地切断二苯并噻吩（DBT）中的C—S键,沿4S途径代谢,生成二羟联苯。实验证明,以2％的接种量脱除50μg／mL DBT底物中的硫效果最佳。在此条件下,适宜菌株生长和脱硫的碳源为葡萄糖,氮源为硝酸铵,初始pH为8.2,生长温度为30℃,15mmol／L的硫酸根离子能使其丧失脱硫能力。在上述适宜条件下,培养72 h后DBT中34.04%的硫被脱除。     

红球菌DS—3脱除二苯并噻吩中有机硫的性能初探

从孤岛油田分离到一株红球菌(Rhodococcus sp.)DS—3,能专一地切断二苯并噻吩(DBT)中的C－S键,沿4S途径代谢,生成二羟联苯。实验证明,以2％的接种量脱除50μｇ／mL DBT底物中的硫效果最佳。在此条件下,适宜菌株生长和脱硫的碳源为葡萄糖,氯源为硝酸铵,初始PH为8．2,生长温度为30℃,15mmol/L的硫酸根离子能使其丧失脱硫能力。在上述适宜条件下,培养72h后DBT中34．04％的硫被脱除。     

Microbial transformations in a cyclodextrin medium. Part 3. Cholesterol oxidation by Rhodococcus erythropolis

An intensive and systematic investigation of the oxidation of cholesterol (CL) to cholest-4-en-3-one (CN) by Rhodococcus erythropolis was undertaken in the presence of natural and chemically modified cyclodextrins (CDs) in a stirred bioreactor. The biotransformation was found to be strongly affected by the mode of addition of the natural CDs. While simultaneous addition of CL with either - or -CD led to a limited enhancement effect, the microbial oxidation of - and -CD complexes of CL was totally inhibited. In contrast, the alkylated CDs- dimethyl-, trimethyl- and hydroxypropyl--CD exhibited a remarkable enhancement of the microbial oxidation, irrespective of their mode of addition. The performance of the alkylated CDs was interpreted in the light of the measured phase solubility diagrams of CL and CN. It was thus shown that unlike the low solubilising power of hydroxypropyl--CD, dimethyl- and trimethyl--CD at 90 mm each, dissolved 9.3 and 8.7 g/l of CL and CN, respectively. Further investigation focused on the formation of CD complexes with CL and CN, analysed by X-ray powder diffractometry, differential scanning calorimetry and ¹H-nuclear magnetic resonance. It was thus shown that -CD forms a 2:1 CD:CL and CD:CN water-insoluble complexes. A mechanism of the biotransformation in homogeneous and heterogeneous CD media was presented while suggesting a direct interaction of the CD-substrate complex with microbial cells. Correspondence to: R. Bar     

Degradation of aromatic amino acids by Rhodococcus erythropolis

A Gram-positive Rhodococcus erythropolis strain S1 was shown to assimilate aromatic amino acids such as L-phenylalanine, L-tyrosine, L-tryptophan, D-phenylalanine, D-tyrosine and D-tryptophan, which were utilized not only as the sole carbon source but also as a suitable nitrogen source. The highest growth on these aromatic amino acids occurred at a temperature of 30°C. L-Phenylalanine, L-tyrosine and L-tryptophan degradative pathways would appear to be independent, and to be induced alternatively. The strain S1 also showed the ability to assimilate peptides which consisted of only L-phenylalanine and L-tyrosine.     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

Biotransformation of heterocyclic dinitriles by Rhodococcus erythropolis and fungal nitrilases

2,6-Pyridinedicarbonitrile (1a) and 2,4-pyridinedicarbonitrile (2a) were hydrated by Rhodococcus erythropolis A4 to 6-cyanopyridine-2-carboxamide (1b; 83% yield) and 2-cyanopyridine-4-carboxamide (2b; 97% yield), respectively, after 10 min. After 118 h, the intermediates 1b or 2b were transformed into 2,6-pyridinedicarboxamide (1c; 35% yield) and 2,6-pyridinedicarboxylic acid (1d; 60% yield) or 2-cyanopyridine-4-carboxylic acid (2c; 64% yield), respectively. The nitrilase from Fusarium solani afforded cyanocarboxylic acids 1e and 2c after 118 h (yields 95 and 62%, respectively). 3,4-Pyridinedicarbonitrile (3a) and 2,3-pyrazinedicarbonitrile (4a) were inferior substrates of nitrile hydratase and nitrilase.     

Haloalkane hydrolysis by Rhodococcus erythropolis cells: comparison of conventional aqueous phase dehalogenation and nonconventional gas phase dehalogenation

Biofiltration of air polluted by volatile organic compounds is now recognized by the industrial and research communities as an effective and viable alternative to standard environmental technologies. Whereas many studies have focused on solid/liquid/gas biofilters, there have been fewer reports on waste air treatment using other biological processes, especially in a solid/gas biofilter. In this study, a comparison was made of the hydrolysis of halogenated compounds (such as 1-chlorobutane) by lyophilized Rhodococcus erythropolis cells in a novel solid/gas biofilter and in the aqueous phase. We first determined the culture conditions for the production of R. erythropolis cells with a strong dehalogenase activity. Four different media were studied and the amount of 1-chlorobutane was optimized. Next, we report the possibility to use R. erythropolis cells in a solid/gas biofilter in order to transform halogenated compounds in corresponding alcohols. The effect of experimental parameters (total flow into the biofilter, thermodynamic activity of the substrates, temperature, carbon chain length of halogenated substrates) on the activity and stability of lyophilized cells in the gas phase was determined. A critical water thermodynamic activity (a(w)) of 0.4 is necessary for the enzyme to become active and optimal dehalogenase activity for the lyophilized cells is obtained for an a(w) of 0.9. A temperature of reaction of 40 degrees C represents the best compromise between stability and activity. Activation energy of the reaction was determined and found equal to 59.5 KJ/mol. The pH effect on the dehalogenase activity of R. erythropolis cells was also studied in the gas phase and in the aqueous phase. It was observed that pH 9.0 provided the best activity in both systems. We observed that in the aqueous phase R. erythropolis cells were less sensitive to the variation in pH than R. erythropolis cells in the gas phase. Finally, the addition of volatile Lewis base (triethylamine) in the gaseous phase and the action of the lysozyme in order to permeabilize the cells was found to be highly beneficial to the effectiveness of the biofilter.     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Molecular Genetic Markers for Identification of Rhodococcus erythropolis and Rhodococcus qingshengii

Microbiology - The application of restriction analysis of amplification products of the genes rpoC and alkB, encoding the synthesis of the DNA-dependent RNA polymerase β'-subunit and...     

Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1

A dibenzothiophene (DBT)-degrading bacterium, Rhodococcus erythropolis D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT → DBT sulfone → 2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBT-desulfurizing ability than other microorganisms reported previously.     

Kinetics of the degradation of aliphatic hydrocarbons by the bacteria Rhodococcus ruber and Rhodococcus erythropolis

Consumption of aliphatic hydrocarbons by the bacteria Rhodococcus ruber Ac-1513-D and Rhodococcus erythropolis Ac-1514-D grown on mixed n-alkanes and diesel fuel was studied. Consumption of diesel fuel hydrocarbons by the strains was less intense in comparison with the n-alkane mixture. The strains showed differences in growth rate and consumption of the substrates, which suggests that they possess different mechanisms of hydrocarbon uptake.