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.     

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.     

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.

     

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.     

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.     

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.     

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.     

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.     

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.     

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.     

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.     

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.     

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.     

Montmorillonite K-10 catalyzed cyclization of N-ethoxycarbonyl-N′-arylguanidines: Access to pyrimido[4,5-c]carbazole and pyrimido[5,4-b]indole derivatives

Two new heterocycles, pyrimido[4,5-c]carbazole and pyrimido[5,4-b]indole, were prepared in three steps from 3-aminocarbazole and 3-aminoindole, respectively. The key Friedel–Crafts intramolecular cyclization was realized under microwave irradiation using montmorillonite K-10 clay as a catalyst. The pyrimido[4,5-c]carbazole derivative shows significant micromolar IC₅₀ against cancer cell lines. Unlike similar carbazole and indolocarbazole compounds, the molecule does not interfere with topoisomerase activity.     

Anti-inflammatory and antiproliferative prenylated carbazole alkaloids from Clausena vestita

Twelve prenylated carbazole alkaloids, containing a novel prenylated carbazole alkaloid, named as clausevestine (1), and 11 known prenylated carbazole alkaloids (2–12), were isolated and identified from the stems and leaves of Clausena vestita, which is a Chinese endemic plant. The chemical structure of 1 was established by means of comprehensive spectroscopic data analyses and the known compounds were determined via comparing their NMR and MS data as well as optical rotation values with those reported in literature. Especially, clausevestine (1) is an unusual prenylated carbazole alkaloid possessing an unprecedented carbon skeleton holding 20 carbon atoms. The anti-inflammatory effects and antiproliferative activities of those isolated prenylated carbazole alkaloids were tested. Prenylated carbazole alkaloids 1–12 displayed remarkable inhibitory effects on NO (nitric oxide) production with IC₅₀ values equivalent to that of the positive control (hydrocortisone). Meanwhile, prenylated carbazole alkaloids 1–12 exhibited remarkable antiproliferative activities against diverse human cancer cell lines in vitro holding the IC₅₀ values ranging from 0.32 ± 0.04 to 18.76 ± 0.18 µM. These findings indicate that these prenylated carbazole alkaloids possessing remarkable anti-inflammatory effects and antiproliferative activities could be meaningful to the discovery of new anti-inflammatory and anti-tumor candidate drugs.     

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.     

Heptazolicine,a carbazole alkaloid from Clausena heptaphylla

A new carbazole alkaloid designated as heptazolicine has been isolated from the roots of Clausena heptaphylla. On the basis of spectral and chemical evidence it has been identified as [2,2-dimethyl-3,4-dihydropyrano-(5, 6-a)]-3-formyl-8-hydroxy-carbazole.     

Design,synthesis and evaluation of carbazole derivatives as potential antimicrobial agents

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5–2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.     

A new dimeric carbazole alkaloid from Murraya koenigii (L.) leaves with α-amylase and α-glucosidase inhibitory activities

A new dimeric carbazole alkaloid, 3,3′,5,5′,8-pentamethyl-3,3′-bis(4-methylpent-3-en-1-yl)-3,3′,11,11′-tetrahydro-10,10′-bipyrano[3,2-a]carbazole, was isolated from the hexane extract of leaves of Murraya koenigii (L.) Sprengel. (Family: Rutaceae). The structure was elucidated based on ¹³C and ¹H NMR, High-Resolution Mass Spectrometry (HRMS), and 2D NMR data. The in vitro antidiabetic activity of the new dimer was investigated in terms of α-amylase and α-glucosidase enzyme inhibition assays. The dimer exhibited significant α-amylase inhibitory activity (IC₅₀ = 30.32 ± 0.34 ppm) and α-glucosidase inhibitory activity (IC₅₀ = 30.91 ± 0.36 ppm).