EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.EDTA is a synthetic chelating agent that has a variety of uses in cleaners, water treatment plants, metal processing, and paper bleaching (41). It can cause mobilization of radionuclides and heavy metals (8,28). Such mobilization increases the exposure of humans to toxic heavy metals and radionuclides. EDTA is not removed by conventional sewage treatment procedures and is recalcitrant in the environment (1, 44). The removal of EDTA can occur via photodegradation of EDTAFe(III) in surface waters (17, 18). Noncomplexed EDTA or EDTA complexed with other metals is not sensitive to photodegradation (24, 30). Although EDTA is recalcitrant, it can be degraded in the environment (7, 26). Belly et al. (4) observed slow degradation of EDTA in an aerated lagoon. Tiedje (45, 46) and Bolton et al. (7) reported slow biodegradation of EDTA in sediments and soils. Three pure cultures of microorganisms have been isolated that are able to degrade EDTA under aerobic conditions: the gram-negative bacterium BNC1 (33, 34), Agrobacterium sp. strain ATCC 55002 (22), and strain DSM 9103 (51).The EDTA monooxygenase has been purified and characterized in strains BNC1 and DSM 9103. In BNC1, an EDTA monooxygenase oxidizes EDTA to ethylenediaminetriacetate (ED3A) and glyoxylate (19,36). In DSM 9103, a similar enzyme oxidizes EDTA to ED3A and then to ethylenediaminediacetate (EDDA) (51). Both EDTA monooxygenases are reduced flavin mononucleotide (FMNH 2 )-utilizing monooxygenases that rely on NAD(P)H:flavin mononucleotide (FMN) oxidoreductases to supply FMNH 2 . However, the genes encoding EDTA-degrading enzymes have not been cloned and sequenced. In ...

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Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA-and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA-and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O 2 . The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent K m for IDA was 4.0 mM with a k cat of 5.3 s ؊1 . When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.Synthetic chelating agents are used in large quantities for a variety of applications in nuclear waste processing, household detergents, water treatments, descaling boilers, and removing the precipitation of sparingly soluble salts (4,23,27,33,38). Aminopolycarboxylic acids and their salts, primarily EDTA, diethylenetriaminepentaacetate, and nitrilotriacetate (NTA), are by far the most dominant group of substances used as chelating agents worldwide. The annual sales of EDTA, NTA, and diethylenetriaminepentaacetate in Europe were 32,550, 18,600, and 14,000 tons in 1997 (27). The environmental disposal of EDTA and NTA can have undesirable consequences. Chelating agents form soluble complexes with radionuclides or heavy metals, increasing their mobility in subsurface environments (9). The mobilized radionuclides and toxic heavy metals can be directly consumed by humans or accumulated by plants and transferred to humans through the food chain, causing health problems.Microbial degradation of chelating agents may decrease the mobilization of radionuclides and heavy metals in the environment. Several enrichment cultures have been reported to mineralize EDTA under strictly aerobic conditions (28,29,40). Three EDTA-degrading microorganisms have been isolated: an Agrobacterium sp. (19), the bacterial strain DSM 9103 (47), and the bacterial strain BNC1 (28). There are also several microorganisms that can use NTA as a sole so...

CardioARM, a highly flexible “snakelike” medical robotic system (Medrobotics, Raynham, MA), has been developed to allow physicians to view, access, and perform complex procedures intrapericardially on the beating heart through a single-access port. Transthoracic epicardial catheter mapping and ablation has emerged as a strategy to treat arrhythmias, particularly ventricular arrhythmias, originating from the epicardial surface. The aim of our investigation was to determine whether the CardioARM could be used to diagnose and treat ventricular tachycardia (VT) of epicardial origin. Animal and clinical studies of the CardioARM flexible robot were performed in hybrid surgical–electrophysiology settings. In a porcine model study, single-port pericardial access, navigation, mapping, and ablation were performed in nine animals. The device was then used in a small, single-center feasibility clinical study. Three patients, all with drug-refractory VT and multiple failed endocardial ablation attempts, underwent epicardial mapping with the flexible robot. In all nine animals, navigation, mapping, and ablation were successful without hemodynamic compromise. In the human study, all three patients demonstrated a favorable safety profile, with no major adverse events through a 30-day follow-up. Two cases achieved technical success, in which an electroanatomic map of the epicardial ventricle surface was created; in the third case, blood obscured visualization. These results, although based on a limited number of experimental animals and patients, show promise and suggest that further clinical investigation on the use of the flexible robot in patients requiring epicardial mapping of VT is warranted.Electronic supplementary materialThe online version of this article (doi:10.1007/s11701-012-0343-6) contains supplementary material, which is available to authorized users.

Hydroxyquinol, a common metabolite of aromatic compounds, is readily auto-oxidized to hydroxyquinone. Enzymatic activities that metabolized hydroxyquinone were observed from the cell extracts of Sphingobium chlorophenolicum ATCC 39723. An enzyme capable of transforming hydroxyquinone was partially purified, and its activities were characterized. The end product was confirmed to be 2,5-dihydroxyquinone by comparing UV/Vis absorption spectra, electrospray mass spectra, and gas chromatography-mass spectra of the end product and the authentic compound. We have proposed that the enzyme adds a H2O molecule to hydroxyquinone to produce 2,5-dihydroxycyclohex-2-ene-1, 4-dione, which spontaneously rearranges to 1, 2,4,5-tetrahydroxybenzene. The latter is auto-oxidized by O2 to 2,5-dihydroxyquinone. The proposed pathway was supported by the overall reaction stoichiometry. Thus, the transformation of hydroxyquinol to 2,5-dihydroxyquinone involves two auto-oxidation of quinols and one enzymatic reaction catalyzed by a hydratase. The specific enzymatic step did not require O2, further supporting the assignment as a hydratase. To our knowledge, this is the first identification of a quinone hydratase, enhancing the knowledge on microbial metabolism of hydroxyquinone and possibly leading to the development of enzymatic method for the production of 2,5-dihydroxyquinone, a widely used chemical in various industrial applications.