Simultaneous Biodetoxification of S, N, and O Pollutants by Engineering of a Carbazole-Degrading Gene Cassette in a Recombinant Biocatalyst

The gene cassette encoding enzymes responsible for degrading carbazole to anthranilic acid was introduced into a dibenzothiophene degrader. The resultant strain, Rhodococcus erythropolis XPDN, could simultaneously transform the model pollutants dibenzothiophene, carbazole, and dibenzofuran to nontoxic metabolites and may have an application potential for bioremediation.     

Selective biodegradation of S and N heterocycles by a recombinant Rhodococcus erythropolis strain containing carbazole dioxygenase

The carbazole dioxygenase genes were introduced into a dibenzothiophene degrader. The recombinant Rhodococcus erythropolis SN8 was capable of efficiently degrading dibenzothiophene and carbazole simultaneously. SN8 could also degrade various alkylated derivatives of carbazole and dibenzothiophene in FS4800 crude oil by just a one-step bioprocess.     

Selective Biodegradation of S and N Heterocycles by a Recombinant Rhodococcus erythropolis Strain Containing Carbazole Dioxygenase

The carbazole dioxygenase genes were introduced into a dibenzothiophene degrader. The recombinant Rhodococcus erythropolis SN8 was capable of efficiently degrading dibenzothiophene and carbazole simultaneously. SN8 could also degrade various alkylated derivatives of carbazole and dibenzothiophene in FS4800 crude oil by just a one-step bioprocess.     

Oxidation of carbazole, N-ethylcarbazole, fluorene, and dibenzothiophene by the laccase of Coriolopsis gallica

Purified laccase from Coriolopsis gallica UAMH8260 oxidized carbazole, N-ethylcarbazole, fluorene, and dibenzothiophene in the presence of 1-hydroxybenzotriazole and 2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as free radical mediators. Susceptibility to laccase oxidation appears related to the ionization potential (IP) of the substrate: compounds with an IP above 8.52, dibenzofuran (IP = 8.77) and benzothiophene (IP = 8.73) were not attacked. Carbazole (IP = 7.68) was the most sensitive to oxidation with >99% transformed with 10 milliunits of laccase after 1 h, though most reactions were carried out for 18 h. 9-Fluorenone was identified as the product of fluorene (IP = 8.52) oxidation, and dibenzothiophene sulfone from dibenzothiophene (IP = 8.44). Although carbazole and N-ethylcarbazole were both completely removed within 18 h, no oxidation or condensation metabolites were detected. This investigation is the first to report the oxidation of dibenzothiophene, carbazole, and N-ethylcarbazole by laccase.     

Biodegradation of Carbazole and Dibenzothiophene by Bacteria Isolated from Petroleum-Contaminated Sites

This study reports the isolation of bacterial cultures, capable of selective removal of nitrogen and sulfur from carbazole and dibenzothiophene, respectively. The isolates utilizing carbazole were found to be suitable for biorefining. These were designated as P10 and P11, and were identified as Pseudomonas sp. Growing cells of P10 and P11 could utilize 77% carbazole in 250 and 120 h, respectively. Isolates showing utilization of dibenzothiophene were not suitable for biorefining industry. Results suggest these Pseudomonas isolates may be useful in petroleum biorefining for the selective removal of organically bound nitrogen from petroleum.     

Bacterial metabolism of fluorene, dibenzofuran, dibenzothiophene, and carbazole

Fluorene and its three heteroatomic analogs, dibenzofuran, dibenzothiophene, and carbazole, are environmental contaminants in areas impacted by spills of creosote. In addition, dibenzofuran has been used as an insecticide, and it is formed from the photolysis of chlorinated biphenyl ethers. Many biodegradation studies of dibenzofuran have considered it as a model for chlorinated dibenzofurans, which are of greater environmental concern. This paper reviews the bacterial degradation of fluorene and its analogs. These compounds are susceptible to three different modes of initial oxidation: (i) the naphthalene-like attack, in which one of the aromatic rings is oxidized to a dihydrodiol; (ii) an angular dioxygenase attack, in which the carbon bonded to the methylene group in fluorene or to the heteroatoms in the analogs, and the adjacent carbon in the aromatic ring are both oxidized; and (iii) the five-membered ring attack, in which the methylene carbon atom in fluorene or the sulfur atom in dibenzothiophene is oxidized. The metabolites, enzymology, and genetics of these transformation are summarized. Literature data are presented, indicating that the electronegativity of the atom connecting the two aromatic rings influences the attack of the angular dioxygenase. In dibenzofuran and carbazole, the connecting atoms, O and N respectively, have high electronegativities, and these compounds serve as substrates for angular dioxygenases. In contrast, the connecting atoms in dibenzothiophene and fluorene, S and C respectively, have lower electronegativities, and these atoms must be oxidized before the angular dioxygenases attack these compounds.     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Biodegradation of carbazole in oil/water biphasic system by a newly isolated bacterium Klebsiella sp. LSSE-H2

A novel Klebsiella sp. strain LSSE-H2 (CGMCC No. 1624) was isolated from dye-contaminated soil based on its ability to metabolize carbazole as a sole source of carbon and nitrogen. This strain efficiently degraded carbazole from either aqueous and biphasic aqueous–organic media, displaying a high denitrogenation activity and a high level of solvent tolerance. LSSE-H2 could completely degrade 12 mmol/L carbazole after 56 h of cultivation. The co-culture of LSSE-H2 and Pseudomonas delafieldii R-8 strains can degrade approximately 92% of carbazole (10 mmol/L) and 94% of dibenzothiophene (3 mmol/L) from model diesel in 12 h.     

The strategy of strain selection for a mixed culture performing rapid conversion of a mixture of polyaromatic compounds

The strain Sphingomonas sp. VKM V-2434 converts the mixture of seven polyaromatic compounds (PACs): fluorene, dibenzothiophene, carbazole, phenanthrene, anthracene, fluoranthene, and pyrene. The effect of each of the above PACs on the rate of mixture conversion was determined. The following two strains, which utilize the substances inhibiting the studied process, were added to the culture: strain FON-11 utilizing 9-fluorenone (fluorene metabolite) and strain CBZ-21 utilizing carbazole. In the case of the mixed culture of three strains, conversion rates were 1.5 and 1.2–3.8 times higher for the PAC mixture and its individual components, respectively, than the rates for Sphingomonas sp. VKM V-2434 monoculture. The degree of degradation of PAC conversion products increased from 32 to 44%. The rate of PAC conversion by the mixed culture exceeded the sum of conversion rates for the individual component strains; this cooperative effect was particularly marked for anthracene and pyrene.     

Dibenzofuran,dibenzothiophene and N-methyl carbazole tethered 2-aminothiazoles and their cinnamamides as potent inhibitors of Mycobacterium tuberculosis

Herein described the design, synthesis and antitubercular evaluation of novel series of dibenzofuran, dibenzothiophene and N-methyl carbazole tethered 2-aminothiazoles and their cinnamamide analogs. One pot condensation of N-methyl carbazole, dibenzofuran and dibenzothiophene methyl ketones with thiourea in the presence of Iodine and CuO gave respective 2-aminothiazoles 4–6 in very good yields. Aminothiazoles were further coupled with substituted cinnamic acids using acid-amine coupling conditions to give desired cinnamamide analogs 8a–e, 9a–e and 10a–e. All the newly synthesized compounds were fully characterized by their NMR and mass spectral analysis. In vitro screening of new derivatives against Mycobacterium tuberculosis H37Rv (Mtb) resulted 8c, 10d and 10e (MIC: 0.78?µg/mL) and 2-aminothiazoles 5 and 6 (MIC: 1.56?µg/mL) as potent compounds with lower cytotoxicity profile.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Ligand recognition by chloroperoxidase using molecular interaction fields and quantum chemistry calculations

We recently reported that chloroperoxidase (CPO) from Caldariomyces fumago showed atypical kinetic behavior for the oxidation of 4,6 dimethyl dibenzothiophene (DMDBT). In this work, we undertake the theoretical study of DMDBT docking into CPO's active site, in order to clarify its binding capacity and affinity using molecular interaction fields and quantum mechanical procedure. The results revealed that CPO has two substrate binding sites with similar affinities for DMDBT. This finding suggests that the atypical kinetic behavior of CPO for the oxidation of DMDBT might be due to the simultaneous binding of two DMDBT molecules during its reaction cycle. Finally, we extend these results to carbazole, a nitrogen-containing PAH, through experimental and theoretical studies.     

Cometabolic Degradation of Dibenzofuran and Dibenzothiophene by a Newly Isolated Carbazole-Degrading Sphingomonas sp. Strain

A carbazole-utilizing bacterium was isolated by enrichment from petroleum-contaminated soil. The isolate, designated Sphingomonas sp. strain XLDN2-5, could utilize carbazole (CA) as the sole source of carbon, nitrogen, and energy. Washed cells of strain XLDN2-5 were shown to be capable of degrading dibenzofuran (DBF) and dibenzothiophene (DBT). Examination of metabolites suggested that XLDN2-5 degraded DBF to 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienic acid and subsequently to salicylic acid through the angular dioxygenation pathway. In contrast to DBF, strain XLDN2-5 could transform DBT through the ring cleavage and sulfoxidation pathways. Sphingomonas sp. strain XLDN2-5 could cometabolically degrade DBF and DBT in the growing system using CA as a substrate. After 40 h of incubation, 90% of DBT was transformed, and CA and DBF were completely removed. These results suggested that strain XLDN2-5 might be useful in the bioremediation of environments contaminated by these compounds.     

Initial Oxidation Products in the Metabolism of Pyrene, Anthracene, Fluorene, and Dibenzothiophene by the White Rot Fungus Pleurotus ostreatus

The initial metabolites in the degradation of pyrene, anthracene, fluorene, and dibenzothiophene by Pleurotus ostreatus were isolated by high-pressure liquid chromatography and characterized by UV-visible, gas-chromatographic, mass-spectrometric, and (sup1)H nuclear magnetic resonance spectral techniques. The metabolites from pyrene, dibenzothiophene, anthracene, and fluorene amounted to 45, 84, 64, and 96% of the total organic-solvent-extractable metabolites, respectively. Pyrene was metabolized predominantly to pyrene trans-4,5-dihydrodiol. Anthracene was metabolized predominantly to anthracene trans-1,2-dihydrodiol and 9,10-anthraquinone. In contrast, fluorene and dibenzothiophene were oxidized at the aliphatic bridges instead of the aromatic rings. Fluorene was oxidized to 9-fluorenol and 9-fluorenone; dibenzothiophene was oxidized to the sulfoxide and sulfone. Circular dichroism spectroscopy revealed that the major enantiomer of anthracene trans-1,2-dihydrodiol was predominantly in the S,S configuration and the major enantiomer of the pyrene trans-4,5-dihydrodiol was predominantly R,R. These results indicate that the white rot fungus P. ostreatus initially metabolizes polycyclic aromatic hydrocarbons by reactions similar to those previously reported for nonligninolytic fungi. However, P. ostreatus, in contrast to nonligninolytic fungi, can mineralize these polycyclic aromatic hydrocarbons. The identity of the dihydrodiol metabolites implicates a cytochrome P-450 monooxygenase mechanism.     

Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.     

Oxygenation Reactions of Various Tricyclic Fused Aromatic Compounds Using Escherichia coli and Streptomyces lividans Transformants Carrying Several Arene Dioxygenase Genes

Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and ¹H and ¹³C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-dihydrophenanthridine, cis-9,10-dihydroxy-9,10-di-hydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.     

Biodegradation of dioxin by a newly isolated Rhodococcus sp. with the involvement of self-transmissible plasmids

A newly isolated Rhodococcus sp. strain p52 could aerobically utilize dibenzofuran as the sole source of carbon and energy, and completely remove dibenzofuran at 500 mg?l^?1 within 48 h. The strain metabolizes dibenzofuran by initial angular dioxygenation to yield 2,2′,3-trihydroxybiphenyl. Strain p52 could also remove 70 % of 100 mg?l^?1 2-chlorodibenzofuran within 96 h and could metabolize a variety of aromatic compounds, namely dibenzo-p-dioxin, 2,8-dichlorodibenzofuran, dibenzothiophene, biphenyl, naphthalene, fluorene, phenanthrene, anthracene, carbazole, indole, xanthene, phenoxathiin, xanthone, and 9-fluorenone. Two distinct gene clusters encoding angular dioxygenases (DbfA and DfdA) were amplified and sequenced. The dbfA and dfdA gene clusters are located on two circular plasmids, pDF01 and pDF02, respectively. Both plasmids are self-transmissible; that is, they can transfer to the Gram-positive bacterium Bacillus cereus by conjugation.     

Diffusion and partitioning of a pesticide, lindane, into phosphatidylcholine bilayers. A new fluorescence quenching method to study chlorinated hydrocarbon-membrane interactions.

Chlorinated hydrocarbons, such as the pesticide lindane (gamma-hexachlorocyclohexane), quench the fluorescence of carbazole. The observed quenching is a result of the molecular contacts which occur upon diffusional collisions. Because the amount of quenching depends upon the collisional frequency between carbazole and pesticide, this phenomenon provides a measure of both the diffusional rate of lindane and its local concentration. The carbazole fluorophore is localized within phosphatidylcholine bilayers by cosonicating the lipid with a newly synthesized phospholipid, beta-(11-(9-carbazole)-undecanoyl)-L-alpha-phosphatidylcholine. Using this probe in dimyristoyl-L-alpha-phosphatidylcholine vesicles, and the above mentioned quenching phenomena, we determined the lindane diffusion rate within the bilayer to be 5.7.10-7 cm2/s at 37 degrees C. Measurement of the apparent quenching constant at various dimyristoyl phosphatidylcholine concentrations yielded a lipid-water partition coefficient for lindane of 9500, which is in agreement with the value of 8980 obtained by our equilibrium dialysis experiments. Vesicles of dimyristoyl-L-alpha-phosphatidylcholine become saturated with lindane at a pesticide to lipid molar ratio of approx. 0.28. These results demonstrate the possibility of using the quenching of carbazole fluorescence to investigate the transport and partitioning of pesticides within biological membranes. This ability should prove useful in studies of the interactions of chlorinated hydrocarbons with cell membranes.     

Mukonal,a probable biogenetic intermediate of pyranocarbazole alkaloids from murraya koenigii

Mukonal, a carbazole alkaloid has been isolated from Murraya koenigii. The structure of the compound has been established as 2-hydroxy-3-formyl carbazole based on physical (UV, IR, ¹H NMR, ¹³C NMR and mass spectrometry) and chemical transformations.