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1.
A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO
2-chloroethanol
- DCPIP
2,6-dichlorophenolindophenol
- FPLC
fast protein liquid chromatography
- PAGE
polyacrylamide gelelectrophoresis
- PES
phenazine ethosulfate
- PMS
phenazine methosulfate
- PQQ
pyrroloquinoline quinone 相似文献
2.
Pseudomonas cepacia MBA4 able to utilize monobromoacetic acid as a sole source of carbon and energy was isolated from soil by enrichment culture. In batch culture the ability to utilize the substrate was conferred by a single halidohydrolase-type dehalogenase which demonstrated a high activity towards the enrichment substrate. The purified enzyme, designated as dehalogenase IVa by activity-stain polyacrylamide gel electrophoresis, had a relative molecular weight of 45,000 and was comprised of two electrophoretically identical subunits with relative molecular weights of 23,000. Dehalogenase IVa demonstrated isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The significance of activity-stain polyacrylamide gel electrophoresis in characterizing dehalogenases and their ubiquitous distribution among bacterial genera are discussed.Abbreviations MCA
Monochloroacetic acid
- DCA
dichloroacetic acid
- MBA
monobromoacetic acid
- 2MPCA
2-monochloropropionic acid
- 2MBPA
2-monobromopropionic acid 相似文献
3.
Andrew W. Thomas Jay Lewington Steve Hope Andrew W. Topping Andrew J. Weightman J. Howard Slater 《Archives of microbiology》1992,158(3):176-182
Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids — HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. the mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment. 相似文献
4.
Abstract: A limited number of halogenated aliphatic compounds can serve as a growth substrate for aerobic microorganisms. Such cultures have (specifically) developed a variety of enzyme systems to degrade these compounds. Dehalogenations are of critical importance. Various heavily chlorinated compounds are not easily biodegraded, although there are no obvious biochemical or thermodynamic reasons why microorganisms should not be able to grow with any halogenated compound. The very diversity of catabolic enzymes present in cultures that degrade halogenated aliphatics and the occurrence of molecular mechanisms for genetic adaptation serve as good starting points for the evolution of catabolic pathways for compounds that are currently still resistant to biodegradation. 相似文献
5.
Mutants of two strains of Pseudomonas putida expressed two cryptic chloroamidases (C-amidase and Hamidase) and one cryptic dehalogenase (DehII). The mutants were selected on either 2-chloropropionamide (2CPA) or 2-monochloropropionate (2MCPA), developing as papillae in parental colonies growing on a metabolisable support substrate. Mutants expressing C-amidase were selected if 2CPA was utilised as either a carbon or a nitrogen source. H-amidase mutants were selected only if 2CPA was used as a nitrogen source. Growth temperature and pH affected the frequency of papillae production, although different temperatures and pHs did not affect the overall growth characteristics of the parental colonies. Decreasing growth temperature increased the frequency of 2cpa+ papillae formation, but decreased the frequency of 2mcpa+ papillae formation. Low pH (6.0) prevented the formation of 2mcpa+ and 2cpa+ papillae. However, in the case of the 2cpa+ papillae, decreasing the growth temperature also allowed papillae formation at pH 6.0.Abbreviations
CAA
Chloroacetamide
-
2CPA
2-Chloropropionamide
-
DCA
Dichloroacetic acid
-
HAA
Halogenated alkanoic acid
-
2MCPA
2-Monochloropropionic acid 相似文献
6.
Twenty three bacterial isolates either pure or consortium were initially screened on the basis of their ability to degrade
as well as dechlorinate 4 — chlorobenzoic acid (4-CBA). Based on comparative growth response, three pure isolates Pseudomonas putida GVS-4, Pseudomonas aeruginosa GVS-18 and Pseudomonas aeruginosa GWS-19 and a consortium SW-2 was finally selected for further studies. The enzyme studies performed with cell free extracts
revealed that dehalogenase activity was substrate specific with maximum activity at 300 μgml−1 substrate concentration. Catechol 1,2 dioxygenase activity was found to be present in cell free extracts suggesting that
4 — chlorobenzoic acid (4-CBA) is catabolized by ortho-ring cleavage pathway. The dehalogenase enzyme profile showed single
enzyme band in case of GVS-4 (Rm 0.76), GVS-18 (Rm 0.84), GWS −19 (Rm 0.85) and two bands in SW-2 (Rm 0.71 & 0.10). 相似文献
7.
Radice F Orlandi V Massa V Battini V Bertoni G Reineke W Barbieri P 《Applied microbiology and biotechnology》2007,75(5):1111-1118
An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order. 相似文献
8.
Heath E Brown WA Jensen SR Bratty MP 《Journal of industrial microbiology & biotechnology》2006,33(3):197-207
The biodegradation of chlorinated alkanes was studied under oxic conditions with the objective of identifying favorable and
unfavorable intramolecular chlorination sequences with respect to the enzymes studied. Several dehalogenating bacterial strains
were screened for their ability to degrade middle-chain polychlorinated alkanes as well as a commercial mixture. Of the organisms
tested, the most promising was Pseudomonas sp. strain 273, which possesses an oxygenolytic dehalogenase. The effects of carbon chain length (C6–C16), halogen position, and overall chlorine content (14–61% w/w) were examined using both commercially available compounds and
molecules synthesized in our laboratory. The effects of co-substrates, solvents, and inducing agents were also studied. The
results with pure chlorinated alkanes showed that the relative positions of the chlorine atoms strongly influenced the total
amount of dehalogenation achieved. The greatest dehalogenation yields were associated with terminally chlorinated alkanes.
The α- and α,ω-chlorinated compounds yielded similar results. Vicinal chlorination had the most dramatic impact on degradation.
When present on both ends or at the center of the molecule, no dehalogenation was detected. Although partial dehalogenation
of 1,2-dichlorodecane was observed, it was likely due to a combination of β-oxidation and an abiotic mechanism. Cereclor S52
was appreciably dehalogenated in shake flasks only when 1,10-dichlorodecane was present as a co-substrate and after increasing
the oil surface area through mechanical emulsification, demonstrating the importance of abiotic factors in degrading commercial
polychlorinated alkane mixtures. 相似文献
9.
There is a significant need for devices capable of measuring water contaminant concentrations in situ––continuously, rapidly, and without reagents, extraction, or other pretreatment. Toward this goal, we constructed and tested fiber optic biosensors for measurement of 1,2-dichloroethane (DCA) in aqueous solutions. The biocomponent was the haloalkane dehalogenase, DhlA, in whole cells of Xanthobacter autotrophicus GJ10. These cells were immobilized in calcium alginate on the tip of a fiber optic fluoresceinamine-based pH optode. The resulting biosensor could quantify DCA at 11 mg/l and had a linear response up to at least 65 mg/l. Total signal change was reached in 8–10 min, and measurements were reproducible (SE <9%). The sensor’s small size, potential for remote operation, and low cost make it of interest for further development. 相似文献
10.
Mohamed Faraj Abdulgader Edbeib 《Bioremediation Journal》2016,20(2):89-97
Halogenated compounds such as α-halocarboxylix acids (αHAs) are widely liberated into the ecosystem through the prevailing xenobiotic activities that involve the use of herbicides for weed management in the agricultural sector and mass production of various commercial halogenated chemical intermediates. Since such compounds exert stress on the environment owing to their recalcitrance and are not easily degraded, the study aimed to isolate, identify, and characterize dehalogenase-producing bacteria with the purpose of bioremediation. The MX1 bacterium was successfully isolated from seawater samples off the coast of Desaru, Malaysia, using an enrichment technique supplemented with 2,2-dichloropropionic acid (2,2-DCP). Interestingly, the MX1 strain grew best in a 20 mM 2,2-DCP minimal medium as the sole carbon source and illustrated a 44 ± 0.2 h cell-doubling time as well as a 38 μmol Cl?/ml maximum rate of chloride ion release. However, 2,2-DCP–containing medium with concentrations that exceeded 30 mM inhibited the growth of the MX1, possibly attributable to the increased toxicity of the compound on the bacteria. Biochemical examinations and 16S rDNA sequence analysis revealed that the MX1 strain shares high identity to Pseudomonas aeruginosa, and the gene sequence was deposited into GenBank as Pseudomonas aeruginosa MX1 under accession number KP336490. The presence of the putative dehalogenase gene in the MX1 strain was established by polymerase chain reaction (PCR) analysis, which proved the presence of conserved amino acid residues belonging to the Group I dehalogenase. This is the first report detailing a P. aeruginosa strain capable of degrading the recalcitrant 2,2-DCP and its functional amino acid residues. 相似文献