Bicyclic Aromatic Compounds Research Articles

Forensic characterization of mid-range petroleum distillates using light biomarkers

ABSTRACTDue to oil refining, commonly used higher boiling biomarkers for oil-source correlation are absent from mid-range petroleum distillates, while lighter biomarkers are concentrated in such products. This study evaluated 63 diagnostic ratios of light biomarkers such as bicyclic sesquiterpanes, diamondoids, and lighter aromatic compounds using 70 diesel oil samples obtained from three Swedish refineries and local gas stations, mostly over a six-month period in 2015. On the basis of their diagnostic power and partial correlation coefficients, a set of 24 ratios is suggested for oil-source correlation of lighter products. The frequency of false positives for this set was determined to be approximately 0.1%. It should be emphasized that in the event of an oil spill, diesel oils are rapidly influenced by weathering and many of the ratios will be affected.

An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.

La-Activated Bicyclo-oligomerization of Acetylene to Naphthalene

We report the first example of metal-mediated acetylene bicyclopentamerization to form naphthalene in the gas phase. The bicyclic aromatic compound was observed in a complex with La. The La(naphthalene) complex was formed by the reaction of laser-ablated La atoms with acetylene molecules in a molecular beam source and was characterized by mass-analyzed threshold ionization spectroscopy. The bicyclo-oligomerization reaction occurs through sequential acetylene additions coupled with dehydrogenation. Three intermediates in the reaction have been identified: lanthanacyclopropene [La(C2H2)], La(cyclobut-1-en-3-yne) [La(C4H2)], and La(benzyne) [(La(C6H4)]. The metal-ligand bonding in the three intermediates is considerably different from that in the La(naphthalene) complex, as suggested by accurately measured adiabatic ionization energies.

A New Index for the Estimation of the Aromatic Character - VIII

A new aromaticity index is proposed based on the energy of π orbitals D={[π1+Σn2(π1-πn)]0 /[π1 + Σn2(π1-πn)]} x a, where n are the number of occupied π orbitals and a is the number of cycles in the molecule. Calculations have been performed on aromatic and heteroaromatic compounds by using DFT method at B3LYP/6-311G+(d,p) level. This new index has been tested on a large number of 6π electron compounds, on bicyclic and polycyclic aromatic compounds and compared with HOMA and the aromatic stabilization energy (ASE). In all the cases, a good correlation has been found. In the case of HOMA, for monocyclic pentaatomic compounds different correlations was found depending on the nature, the number, and the position of the heteroatoms. Keywords: Aromaticity, DFT, HOMA, ASE, quantification of the aromatic character.

모델 콜타르 유분 중에 함유된 질소고리화합물의 추출에 관한 메탄올과 포름아마이드의 비교

The separation of nitrogen heterocyclic compound (NHC) contained in a model coal tar fraction was compared by the methanol and formamide extraction. The model coal tar fraction comprising four kinds of NHC (NHCs : quinoline, iso-quinoline, indole, quinaldine) and three kinds of bicyclic aromatic compound (BACs : 1-methylnaphthalene, 2-methylnaphthalene, dimethylnaphthalene), biphenyl and phenyl ether was used as a raw material. The aqueous solution of methanol and formamide were used as solvents. A batch-stirred tank was used as the raw material - a solvent contact unit of this work. Independent of the solvent used, the distribution coefficient of NHCs sharply increased by decreasing the initial volume ratio of water to the solvent and increasing the equilibrium operation temperature, whereas, the selectivity of NHCs in reference to BACs decreased. Decreasing the initial volume ratio of solvent to feed resulted in deteriorating distribution coefficients, but the selectivity of NHCs in reference to BAC was almost the constant. The distribution coefficient of NHCs by the methanol extraction was 3∼5 times higher than that of NHCs by the formamide extraction, inversely, the selectivity of NHCs based on BACs by the formamide extraction was 3∼7 times higher than that of NHCs by the methanol extraction. Furthermore, two different solvent extraction methods by adding the extraction processing speed to the balance between solvency and selectivity of NHCs were compared.

추출-증류-결정의 조합에 의한 콜타르 흡수유 중에 함유된 인돌의 고순도 정제

Purification of indole contained in model coal tar absorption oil was examined by extraction-distillation-crystallization combination. The absorption oil consists of nine components such as four kinds of nitrogen heterocyclic compounds (9.2% quinoline, 2.4% iso-quinoline, 4.7% indole, 2.4% quinaldine), three kinds of bicyclic aromatic compounds (14.2% 1-methylnaphthalene, 31.8% 2-methylnaphthalene, 23.5% dimethylnaphthalene), 5.5% biphenyl and 3.3% phenyl ether. 99.5% indole was recovered by combination of formamide extraction-distillation-solute crystallization using n-hexane. Furthermore, the recovery process of indole contained in coal tar absorption oil was studied by using the experimental results obtained by each operation of this work.

9성분계 모델 콜타르 흡수유 중에 함유된 질소고리화합물의 메탄올 추출

9성분계 모델 콜타르 유분 중에 함유된 질소고리화합물(NHC)의 분리를 분배평형에 의해 검토했다. 원료로서는 NHC group (NHCs; quinoline (Q), iso-quinoline (iQ), indole (In), quinaldine(Qu)), 2환 방향족 화합물 group (BACs; 1-methylnaphthalene (1MN), 2-methylnaphthalene (2MN), dimethylnaphthalene (DMN)), biphenyl (Bp)과 phenyl ether (Pe)로 제조한 모델 콜타르 유분을, 용매로서는 메탄올 수용액을 각각 사용했다. 액-액 접촉 장치로서는 회분 교반기를 각각 사용했다. 평형조작 온도가 상승함에 따라 NHCs의 분배계수는 증가했으나, 역으로 BACs를 기준한 NHCs의 선택도는 감소했다. 초기 물/용매의 체적비가 감소함에 따라 BACs를 기준한 NHCs의 선택도는 급격히 감소했으나, NHCs의 분배계수는 증가했다. 일정 조건하에서, 각 group별 분배계수와 BACs를 선택도의 순서는 NHCs > Bp > BACs > Pe와 NHCs > Bp > Pe를 각각 나타냈다. 또한 전체 성분에 대한 분배계수의 순서는 In > iQ = Q > Qu > Bp > 1MN = 2MN > Pe > DMN을 나타냈다. 메탄올 추출에 의해 얻어진 NHCs의 최대 수율과 BACs를 기준한 NHCs의 최대 선택도는 각각 94%, 23이었다. 본 연구를 통해 얻어진 실험적 결과를 이용하여 콜타르 중에 함유된 NHCs의 회수공정을 검토했다. The separation of nitrogen heterocyclic compound (NHC) contained in a model coal tar fraction of nine components system was investigated by distribution equilibrium. The model coal tar fraction comprising NHC group (NHCs; indole (In), quinoline (Q), iso-quinoline (iQ), quinaldine(Qu)), bicyclic aromatic compound group (BACs; 1-methylnaphthalene (1MN), 2-methylnaphthalene (2MN), dimethylnaphthalene (DMN)), biphenyl (Bp) and phenyl ether (Pe) and the aqueous methanol were used as the raw materials and the solvent of this work, respectively. A batch-stirred tank was used as the liquid-liquid contact unit of this work. The distribution coefficient of NHCs increased by increasing the equilibrium operation temperature, whereas the selectivity of NHCs with respect to BACs decreased. Decreasing the initial volume ratio of water to the solvent resulted in deteriorating the selectivity of NHCs in reference to BACs, but improving the distribution coefficients of NHCs. At a fixed experimental condition, the sequence of the distribution coefficient and the selectivity with reference to BACs for each groups was increased in order of NHCs > Bp > BACs > Pe and NHCs > Bp> Pe, respectively. Also, the sequence of the distribution coefficient for entire compounds was in order of In > iQ = Q > Qu > Bp > 1MN = 2MN > Pe > DMN. The maximum yield of NHCs and the selectivity of NHCs based on BACs obtained by methanol extraction were 94 and 23%, respectively. Furthermore, the recovery process for NHCs from coal tar was studied by using the experimental results from this work.

A New Index for the Estimation of the Aromatic Character - II

A new aromaticity index has been proposed based on the energy of π orbital {-[π1+Σ2n(π1-πn)]×103} where n is the number of occupied orbitals. Calculations have been performed on aromatic and heteroaromatic compounds using DFT method at B3LYP/6-311G+(d,p) level. This index has been tested on 6π electron compounds, on bicyclic and polycyclic aromatic compounds and compared with HOMA, the molar magnetic susceptibility (XM), and the aromatic stabilization energy (ASE). In all the cases, a good correlation has been found. Keywords: Aromaticity, DFT, quantification of the aromatic character.

Adsorption of aromatic compounds on porous covalent triazine-based framework

Covalent triazine-based frameworks (CTFs) are an emerging class of polymers whose adsorption properties of organic chemicals are not well understood. The main objective of this work was to evaluate combined effects of the functional groups of aromatic solutes and the triazine structure of a synthesized CTF on adsorption in aqueous solutions. Adsorption of the hydroxyl-, amino-, nitro-, and sulfonate-substituted monocyclic and bicyclic aromatic compounds was generally stronger than their non-substituted, nonpolar counterparts (benzene and naphthalene). When compared with Amberlite XAD-4 resin, one of the most common and widely used polymeric adsorbents, the CTF showed much stronger adsorption toward the polar and/or ionic compounds. To explain the adsorption enhancement of CTF, several specific, non-hydrophobic mechanisms were proposed, including hydrogen bonding (hydroxyl- and amino-substituted compounds), electrostatic attraction (anionized compounds), and π–π electron-donor–acceptor (EDA) interaction (nitroaromatic compounds) with the triazine structure of CTF. The hypothesized mechanisms were further supported by the observed pH dependence of adsorption. Resulting from size exclusion, adsorption of large-size dissolved humic acids on the homogeneous, nanopored (1.2nm in size) CTF was negligible and did not affect adsorption of aromatic solutes. Additional advantages of fast adsorption/desorption kinetics and complete adsorption reversibility made CTF a superior adsorbent for aromatic compounds.

ChemInform Abstract: Biocatalytic Production of Hydroxylated Aromatic and Alicyclic Compounds: Products Derived from Tetralin.

Biological hydroxylation is an area with many opportunities for the production of fine chemicals. Especially for the production of hydroxylated aromatic and alicyclic compounds, the specificity and mildness of biocatalysis can be beneficial. 1,2,3,4-Tetrahydronaphthalene (Tetralin) is a bicyclic aromatic compound, consisting of an aromatic and an alicyclic moiety. The biological oxidation of this compound provides an interesting model reaction, because different sites of initial attack are possible. Eight rains of bacteria that utilize tetralin as sole source of carbon and energy have been teed for the formation and accumulation of interesting oxidation products. Accumulation of oxidation products characteristic of both the oxidation of the aromatic and the alicyclic moiety has been demonstrated. Two rains, a Corynebacterium and an Acinetobacter have been studied in more detail. Corynebacterium C125 starts with initial attack on the aromatic ring by a dioxygenase type of enzyme resulting in the formation of a cis-dihydro diol that is chemically dehydrated to a phenol in an acid environment. Acinetobacter T5 starts with oxidation of the benzylic carbon atom, resulting in the formation of an alcohol, that is further oxidized via a ketone.

Liquid Membrane Permeation of Nitrogen Heterocyclic Compounds Contained in Model Coal Tar Fraction

We investigated the separation of nitrogen heterocyclic compound (NHC) contained in a model coal tar fraction comprising four kinds of NHC [indole (In), quinoline (Q), iso-quinoline (iQ), quinaldine (Qu)], three kinds of bicyclic aromatic compound (BAC) [1-methylnaphthalene (1MN), 2-methylnaphthalene (2MN), dimethylnaphthalene (DMN) mixture with ten structural isomers (DMNs; regarded as one component)], biphenyl (Bp) and phenyl ether (Pe) by liquid membrane permeation (LMP). A batch-stirred tank was used as the permeation unit. An aqueous solution of saponin and n-hexane were used as the liquid membrane and the outer oil phase, respectively. Yield and selectivity of individual NHC was much larger than that of BAC, Bp and Pe. Increasing the initial mass fraction of the saponin to the membrane solution () and the initial volume fraction of O/W emulsion to total liquid in a stirred tank () resulted in deteriorating the yield of individual NHC, but increasing the stirring speed (N) resulted in improving the yield of each NHC. With increasing , the selectivity of each NHC based on DMNs increased. Increasing and N resulted in decreasing the selectivity of individual NHC based on DMNs. At an experimental condition fixed, the sequence of the yield and selectivity in reference to DMNs for each NHC was Q > Qu = iQ > In. Furthermore, we compared LPM method with methanol extraction method in view of the separation efficiency (yield, selectivity) of NHC.

An experimental and kinetic modeling study of the autoignition of α-methylnaphthalene/air and α-methylnaphthalene/ n-decane/air mixtures at elevated pressures

The autoignition of α-methylnaphthalene (AMN), the bicyclic aromatic reference compound for the cetane number (CN), and AMN/ n-decane blends, potential diesel surrogate mixtures, was studied at elevated pressures for fuel/air mixtures in a heated high-pressure shock tube. Additionally, a comprehensive kinetic mechanism was developed to describe the oxidation of AMN and AMN/ n-decane blends. Ignition delay times were measured in reflected shock experiments for Φ = 0.5, 1.0, and 1.5 AMN/air mixtures (CN = 0) for 1032–1445 K and 8–45 bar and for Φ = 1.0 30%-molar AMN/70%-molar n-decane/air (CN = 58) and 70%-molar AMN/30%-molar n-decane/air mixtures (CN = 28) for 848–1349 K and 14–62 bar. Kinetic simulations, based on the comprehensive AMN/ n-decane mechanism, are in good agreement with measured ignition times, illustrating the emerging capability of comprehensive mechanisms for describing high molecular weight transportation fuels. Sensitivity and reaction flux analysis indicate the importance of reactions involving resonance stabilized phenylbenzyl radicals, the formation of which by H-atom abstractions with OH radicals has an important inhibiting effect on ignition.

Impact of dibenzofuran/dibenzo-p-dioxin amendment on bacterial community from forest soil and ring-hydroxylating dioxygenase gene populations

The impact of dibenzofuran (DF) and dibenzo-p-dioxin (DD) on the changes in bacterial community structure and the transition of catabolic genes were studied using forest soil. The bacterial community structure of soil suspensions amended with 1 microg/g of either DF or DD was analyzed by 16S rRNA and functional gene sequencing. To analyze the functional genes in the communities, we targeted a gene sequence that functions as the binding site of Rieske iron sulfur center common to ring-hydroxylating dioxygenases (RHDs) for monocyclic, bicyclic, and tricyclic aromatic compounds. The gene fragments were polymerase chain reaction-amplified from DNAs extracted from soil suspensions spiked with either DF or DD, cloned, and sequenced (70 clones). Bacterial community analysis based on 16S rRNA genes revealed that specific 16S rRNA gene sequences, in particular, phylotypes within alpha-Proteobacteria, increased in the soil suspension amended with DF or DD. RHD gene-based functional community analysis showed that, in addition to two groups of RHD genes that were also detected in unamended soil suspensions, another two groups of RHD genes, each of which is specific to DF- and DD-amended soil, respectively, emerged to a great extent. The DD-specific genotype is phylogenetically distant from any known RHDs. These results strongly suggest that soil microbial community potentially harbors a wide array of organisms having diverse RHDs including those previously unknown, and that they could quickly respond to an impact of contamination of hazardous chemicals by changing the microbial community and gene diversity.

Water‐soluble calix[4]resorcinarenes as chiral NMR solvating agents for bicyclic aromatic compounds

Water-soluble calix[4]resorcinarenes with proline, 3-hydroxyproline, and 4-hydroxyproline substituent groups are evaluated as chiral NMR solvating agents on a series of bicyclic aromatic compounds with naphthyl, indole, dihydroindole, and indane rings. The substrates interact with the calixresorcinarene through insertion of the aromatic ring into the cavity. Most of the substrates are analyzed as cationic species, although one anionic species is analyzed. All of the substrates exhibit enantiomeric discrimination in the 1H-NMR spectrum with one or more of the calixresorcinarenes. In most cases, the hydroxyproline derivatives are more effective at causing enantiodifferentiation than the corresponding proline derivative. Presumably, the hydroxyl group on the proline moieties is involved in interactions with the substituent groups of the substrate that are important in creating chiral recognition. The enantiomeric discrimination in the 1H-NMR spectrum is large enough for many resonances to permit the analysis of enantiomeric purity.

Oxidation of mono- and bicyclic aromatic compounds with hydrogen peroxide catalyzed by titanium silicalites TS-1 and TS-1B

Abstract The hydroxylation of a series of oxygenated aromatic compounds (phenols and ethers) with hydrogen peroxide, catalyzed by titanium silicalite TS-1 and a new catalyst TS-1B, was investigated operating in a biphasic reaction medium. In several cases, the reaction afforded phenols with fairly good selectivity. TS-1B is often a better catalyst than the conventional TS-1, displaying higher selectivity on hydrogen peroxide and also higher shape selectivity. So it appears to have some potential in the direct synthesis of selected phenols, in alternative to the conventional multi-step methods.

2,3-Dihydroxybiphenyl dioxygenase gene was first discovered in Arthrobacter sp. strain PJ3

Bacterium strain PJ3, isolated from wastewater and identified as Arthrobacter sp. bacterium based on its 16S rDNA gene, could use carbazole as the sole carbon, nitrogen and energy source. The genomic library of strain PJ3 was constructed and a positive clone JM109 (pUCW402) was screened out for the expression of dioxygenase by the ability to form yellow ring-fission product. A 2,3-dihydroxybiphenyl dioxygenase (23DHBD) gene of 933 bp was found in the 3360 bp exogenous fragment of pUCW402 by GenSCAN software and BLAST analysis. The phylogenetic analysis showed that 23DHBD from strain PJ3 formed a deep branch separate from a cluster containing most known 23DHBD in GenBank. Southern hybridization confirmed for the first time that the 23DHBD gene was from the genomic DNA of Arthrobacter sp. PJ3. In order to test the gene function, recombinant bacterium BL21 (pETW-8) was constructed to express 23DHBD. The expression level in BL21 (pETW-8) was highest compared with the recombinant bacteria JM109 (pUCW402) and strain PJ3. We observed that 23DHBD was not absolute specific. The enzyme activity was higher with 2,3-dihydroxybiphenyl as a substrate than with catechol. The substrate specificity assay suggested that 23DHBD was essential for cleavage of bi-cyclic aromatic compounds during the course of aromatic compound biodegradation in Arthrobacter sp. strain PJ3.

Genetic and biochemical characterization of the dioxygenase involved in lateral dioxygenation of dibenzofuran from Rhodococcus opacus strain SAO101

Rhodococcus opacus strain SAO101 was shown to degrade on various polycyclic aromatic hydrocarbons such as naphthalene, dibenzofuran (DF), and dibenzo-p-dioxin (DD). One of the unique traits of the strain SAO101 is its ability to oxidize DF compounds by lateral dioxygenation. To clone the lateral dioxygenase gene involved in compound degradation in strain SAO101, we identified a cosmid clone that oxidizes aromatic compounds by using SAO101 genomic DNA. Sequencing analysis revealed that isolated cosmid clone contained ring-hydroxylating dioxygenase genes (narAaAb) with homologies to indene dioxygenase genes of Rhodococcus strain I24 and naphthalene dioxygenase genes of Rhodococcus strain NCIMB12038. The NarAaAb-expressing Rhodococcus cells exhibited broad substrate specificity for bicyclic aromatic compounds and had high ability to degrade dibenzofuran and naphthalene. Metabolite analysis revealed that dihydrodiol compounds were detected as metabolites from dibenzofuran by the NarAaAb-expressing Rhodococcus strain, indicating that dibenzofuran was converted by lateral dioxygenase activity of NarA dioxygenase. Based on reverse transcriptase-polymerase chain reaction analysis, it was found that the narAaAb genes were cotranscribed and that their expression was induced in the presence of aromatic hydrocarbon compounds. It is likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by this strain. Strain SAO101 harbors three huge linear plasmids, pWK301 (1,100 kbp), pWK302 (1,000 kbp), and pWK303 (700 kbp), and the nar genes were found to be located on the pWK301 plasmid.

Uptake of Cycloalkanes and Bicyclic Aromatic Compounds by Ion-Exchange Resins from Aqueous Solutions

The uptakes of cycloalkanes (C5 - C8) and bicyclic aromatic compounds (naphthalene, biphenyl, and azulene) by both strong acid cation (Dowex 50W-X4 and X8) and strong base anion (Dowex 1-X4 and X8) exchange resins have been studied in aqueous solutions at 25 degrees C; their distribution constants (K) have been determined. The relationship between the octanol-water partition coefficient and the resin affinity for solutes has been analyzed. The K values of three aromatic compounds were found to be much higher than expected from the octanol-water partition coefficients. This is probably due to the pi interaction of aromatic rings with the resin matrix. Microscopic observations revealed that azulene molecules are uniformly distributed inside a resin particle.

Separation of Nitrogen Heterocyclic Compounds from Model Coal Tar Fraction by Solvent Extraction

The separation of four kinds of nitrogen heterocyclic compounds (NHCs) from a model mixture comprising NHCs [indole (In), quinoline (Q), iso‐quinoline (iQ), quinaldine (Qu)], three kinds of bicyclic aromatic compounds [BACs; 1‐methylnaphthalene (1MN), 2‐methylnaphthalene (2MN), dimethylnaphthalene (DMN)], biphenyl (Bp) and phenyl ether (Pe) was examined by a solvent extraction. The model mixture used as a raw material of this work was prepared according to the components and compositions contained in coal tar fraction (the temperature ranges of fraction: 240–265°C). The distribution equilibrium between the coal tar fraction and the solvent was measured with eight kinds of solvents. An aqueous solution of methanol, ethanol, iso‐propyl alcohol, N,N‐dimethyl acetamide, DMF, formamide, N‐methylformamide/methanol, and formamide/methanol were used as solvents. An aqueous solution of formamide was found to be suitable for separating NHCs contained in coal tar fraction based on distribution coefficient and selectivity. The effect of operation factors on separating NHCs was investigated by the distribution equilibrium using an aqueous solution of formamide. Increasing the operation temperature and the volume ratio of solvent to feed at initial (S/F)0 resulted in improving the distribution coefficients of each NHC, but increasing the volume fraction of water in the solvent at initial (yw,0) resulted in deteriorating the distribution coefficients of each NHC. With increasing yw,0 and (S/F)0, the selectivities of each NHC in reference to DMN increased. Increasing the operation temperature resulted in decreasing the selectivities of each NHC in reference to DMN. At an experimental condition fixed, the sequence of the distribution coefficient and selectivity in reference to DMN for each NHC was In>iQ>Q>Qu, and also the sequence of the distribution coefficient for each BAC was 1MN>2MN>DMN. The sequence of the distribution coefficient for entire compounds analyzed by this work was In> iQ>Q>Qu>Bp>1MN>2MN>Pe>DMN.

Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving the aromatic C-C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. Based on curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from P. aeruginosa ZD 4-3 able to degrade both single and bicyclic compounds via the meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it had one ORF, which showed a strong amino acid sequence similarity to the known C23Os of mesophilic gram-negative bacteria. The alignment analysis indicated that distinct difference existed between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases cleaving bicyclic aromatic compounds. The heterogenous expression of the pheB gene in Escherichia coli BL21(DE3) demonstrated that this C23O possessed a meta-cleavage activity.