Oxygenases without requirement for cofactors or metal ions

Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.     

Using synthetic chemistry to understand copper protein active sites: a personal perspective

The results of studies performed in the author’s laboratory are surveyed, with particular emphasis on demonstrating the value of a multidisciplinary synthetic modeling approach for discovering new and unusual chemistry helpful for understanding the properties of the active sites of copper proteins or assessing the feasibility of mechanistic pathways they might follow during catalysis. The discussion focuses on the progress made to date toward comprehending the nitrite reductase catalytic site and mechanism, the electronic structures of copper thiolate electron transfer centers, the sulfido-bridged “Cu_Z” site in nitrous oxide reductase, and the processes of dioxygen binding and activation by mono- and dicopper centers in oxidases and oxygenases.     

Characteristics and biotechnological applications of microbial cholesterol oxidases

Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.     

Structure and biogenesis of topaquinone and related cofactors

 The structure of a new biological redox cofactor – topaquinone (TPQ), the quinone of 2,4,5-trihydroxyphenylalanine – was elucidated in 1990. TPQ is the cofactor in most copper-containing amine oxidases. It is produced by post-translational modification of a strictly conserved active-site tyrosine residue. Recent work has established that TPQ biogenesis proceeds via a novel self-processing pathway requiring only the protein, copper, and molecular oxygen. The oxidation of tyrosine to TPQ by dioxygen is a six-electron process, which has intriguing mechanistic implications because copper is a one-electron redox agent, and dioxygen can function as either a two-electron or four-electron oxidant. This review adopts an historical perspective in discussing the structure and reactivity of TPQ in amine oxidases, and then assesses what is currently understood about the mechanism of the oxidation of tyrosine to produce TPQ. Aspects of the structures and chemistry of related cofactors, such as the Tyr-Cys radical in galactose oxidase and the lysine tyrosylquinone of lysyl oxidase, are also discussed. Received: 23 May 1998 / Accepted: 19 October 1998     

The oxygenase-peroxidase theory of Bach and Chodat and its modern equivalents: Change and permanence in scientific thinking as shown by our understanding of the roles of water,peroxide, and oxygen in the functioning of redox enzymes

Alexander Bach was both revolutionary politician and biochemist. His earliest significant publication, “Tsargolod” (“The Tsar of Hunger”), introduced Marxist thought to Russian workers. In exile for 30 years, he moved to study the dialectic of the oxidases. When his theory of oxidases as combinations of oxygenases and peroxidases was developed (circa 1900) the enzyme concept was not fully formulated, and the enzyme/substrate distinction not yet made. Peroxides however were then and remain now significant intermediates, when either free or bound, in oxidase catalyses. The aerobic dehydrogenase/peroxidase/catalase coupled systems which were studied slightly later clarified the Bach model and briefly became an oxidase paradigm. Identification of peroxidase as a metalloprotein, a key step in understanding oxidase and peroxidase mechanisms, postdated Bach’s major work. Currently we recognize catalytic organic peroxides in flavoprotein oxygenases; such organic peroxides are also involved in lipid oxidation and tryptophan radical decay. But most physiologically important peroxides are now known to be bound to transition metals (either Fe or Cu) and formed both directly and indirectly (from oxygen). The typical stable metalloprotein peroxide product is the ferryl state. When both peroxide oxidizing equivalents are retained the second equivalent is held as a protein or porphyrin radical. True metal peroxide complexes are unstable. But often water molecules mark the spot where the original peroxide decayed. The cytochrome c oxidase Fe-Cu center can react with either peroxide or oxygen to form the intermediate higher oxidation states P and F. In its resting state water molecules and hydroxyl ions can be seen marking the original location of the oxygen or peroxide molecule. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1278–1288.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

Site-directed mutagenesis and computational study of the Y366 active site in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> protoporphyrinogen oxidase

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of heme and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX, with FAD as cofactor. Among PPO, Bacillus subtilis PPO (bsPPO) is unique because of its broad substrate specificity and resistance to inhibition by diphenylethers. Identification of the activity of bsPPO would help us to understand the catalysis and resistance mechanisms. Based on the modeling and docking studies, we found that Y366 site in bsPPO was adjacent to substrate and FAD. In order to evaluate the functional role of this site, three mutants Y366A Y366E and Y366H were cloned and kinetically characterized. The efficiency of catalysis for Y366A and Y366H reduced to 10% of the wild-type enzyme’s activity, while Y366E just retained 1%. Y366E shows large resistance (K _i = 153.94 μM) to acifluorfen. Molecular docking was carried out to understand the structure and functional relationship of PPO. The experimental results from the site-directed mutagenesis are consistent with the computational studies. The residue at position 366 is seemed to be responsible for substrate binding and catalysis and involved in herbicide resistance of bsPPO.     

Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea

Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn²⁺ and exhibited high thermostability up to 70°C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.     

Intramolecular electron transfer rate between active-site copper and TPQ in Arthrobacter globiformis amine oxidase

Copper amine oxidases catalyze the oxidative deamination of primary amines operating through a ping-pong bi bi mechanism, divided into reductive and oxidative half-reactions. Considerable debate still exists regarding the role of copper in the oxidative half-reaction, where O₂ is reduced to H₂O₂. Substrate-reduced amine oxidases display an equilibrium between a Cu(II) aminoquinol and a Cu(I) semiquinone, with the magnitude of the equilibrium constant being dependent upon the enzyme source. The initial electron transfer to dioxygen has been proposed to occur from either the reduced Cu(I) center or the reduced aminoquinol cofactor. In order for Cu(I) to be involved, it must be shown that the rate of electron transfer (k _ET) between the aminoquinol and Cu(II) is sufficiently rapid to place the Cu(I) semiquinone moiety on the mechanistic pathway. To further explore this issue, we measured the intramolecular electron transfer rate for the Cu(II) aminoquinol ⇆ Cu(I) semiquinone equilibrium in Arthrobacter globiformis amine oxidase (AGAO) by temperature-jump relaxation techniques. The results presented herein establish that k _ET is greater than the rate of catalysis (k _cat) for the preferred amine substrate β-phenylethylamine at three pH values, thereby permitting the Cu(I) semiquinone to be a viable catalytic intermediate during enzymatic reoxidation in this enzyme. The data show that k _ET is approximately equivalent at pH 6.2 and 7.2, being 2.5 times k _cat for these pH values. At pH 8.2, however, k _ET decreases, becoming comparable to k _cat. Potential reasons for the decreased k _ET at basic pH are presented. The implications of these results in light of a previously published study measuring reoxidation rates of substrate-reduced AGAO are also addressed.     

Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).     

Identification of dioxygenases required for Aspergillus development. Studies of products, stereochemistry, and the reaction mechanism

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-(2)H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Expression of the Renilla reniformis Luciferase Gene in Mammalian Cells

A cDNA encoding the Renilla reniformis luciferase was expressed in simian and murine cells in a transient and stable manner, respectively. Light emission catalyzed by luciferase was detected from transfected cells both in vitro and in vivo. This work establishes the Renilla luciferase gene as a new efficient marker of gene expression in mammalian cells.     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

Evidence for similar biochemical requirements for bioluminescence among the coelenterates

We present evidence that the chemical requirements among all the bioluminescent coelenterates that have been examined are very similar or identical to those already described for Renilla by Cormier and associates. Components required for luminescence in Renilla were also found in a number of bioluminescent coelenterates examined such as Aequorea, Obelia, Cavernularia, Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus and Mnemiopsis. Depending on the organism these include one or more of the following: luciferyl sulfate, luciferase, and luciferin sulfokinase. These isolated components were found to be indistinguishable from those found in Renilla as evidenced by their reactivity in the Renilla bioluminescent system, by the spectral characteristics of the isolated luciferyl sulfates, by the molecular weights of the luciferases, and by the colors of the bioluminescence produced in vitro.     

Enzymatic epoxidation reactions

The oxygenases - enzymes which incorporate molecular oxygen directly into organic molecules - are ubiquitous and of high metabolic significance. These enzymes play crucial roles in the degradation of drugs and foreign substances and in the biosynthesis, interconversion and degradation of amino acids, lipids, porphyrins, vitamins and hormones. Thus, they are centrally involved in the mechanisms of cytotoxicity, mutagenicity, carcinogenicity and tissue necrosis. From the standpoint of enzyme technology, the ability of these enzymes to incorporate molecular oxygen into organic substrates efficiently and selectively is highly enticing, since such reactions are poorly accomplished using conventional chemistry. This review focuses on enzymatic epoxidation reactions, one example of the many chemical transformations catalysed by oxygenases. By way of introduction, an overview of the role of enzymatic epoxidation reactions in the metabolism of polycyclic aromatic hydrocarbons, in steroid biosynthesis and interconversion, and in various other pathways is presented. Following this, enzymatic epoxidation of simple olefins is considered in detail, with emphasis on bacterial systems and discussion of both enzymology and reactivity characteristics. Finally, a number of major issues which must be confronted if complex oxygenase systems are to be utilized in enzyme technology application are briefly discussed. Among these are specialized immobilization techniques, cofactor recycling, problems of enzyme stability, and the intriguing possibility of utilizing mechanistic information in the design of non-enzymatic, chemical model systems which mimic oxygenase catalysis.     

Purification and characterization of extracellular cholesterol oxidase from <Emphasis Type="Italic">Enterobacter</Emphasis> sp.

The objective of this work is to obtain an abundant source of cholesterol oxidases for industrial and medicinal needs. Thirteen bacterial strains that express high level of inducible extracellular cholesterol oxidase (COX) were isolated from carnivore feces. One of these strains, named COX8-9, belonging to the genus Enterobacter, was found to produce the highest level of cholesterol oxidase. COX from strain COX8-9 was purified from the culture supernatant by ultrafiltration followed with two consecutive Q-Sepharose chromatographies at different pH values, and then by Superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The K _m value for oxidation of cholesterol by this enzyme was 1.2 × 10⁻⁴ M, with optimum activity at pH 7.0. Enzymatic activity of COX was enhanced 3-fold in the presence of metal ion Cu²⁺, and the enzyme was stable during long-term aqueous storage under various temperatures, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way. Deping Ye and Jiahong Lei are contributed equally to this work.     

Isolation and Characterization of Dibenzofuran-Degrading <Emphasis Type="Italic">Serratia marcescens</Emphasis> from Alkalophilic Bacterial Consortium of the Chemostat

Alkalophilic bacterial consortium developed by continuous enrichment in the chemostat in presence of 4-chlorosalicylic acid as sole source of carbon and energy contained six bacterial strains, Micrococcus luteus (csa101), Deinococcus radiothilus (csa102), csa103 (Burkholderia gladioli), Alloiococcus otilis (csa104), Micrococcus diversus (csa105), Micrococcus luteus (csa106), identified by the Biolog test method. The strains were tested for utilization of organic compounds in which one of the strains (csa101) had higher potency to utilize dibenzofuran (DF) as sole carbon and energy source identified as Serratia marcescens on the basis of 16S rDNA. The degradation of DF by bacterial strain proceeded through an oxidative route as indicated by 2,2′3-trihydroxybiphenyl, 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid, salicylic acid, and catechol, which was identified by gas chromatography–mass spectrometry.