• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.245-249
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• 2007

Chloroanilines are widely used in the production of dyes, drugs and herbicides. Chloroanilines, however, are considered potential pollutants due to their toxic and recalcitrant properties to humans and other species. With the increase of necessity of bioremediation, this study was conducted to isolate the chloroanilines-degrading bacteria. A bacterium capable of growth on 3,4-dichloroaniline (DCA) was isolated by the 3,4-DCA-containing enrichment culture. The strain KB35B was identified as Pseudomonas sp. and also able to degrade several chloroanilines. The isolated strain showed high level of catechol 2,3-dioxygenase activity in the presence of 3,4-DCA. The activity of catecho1 2,3-dioxygenase was supposed to be ones of the important factors for 3,4-DCA degradation. The activity toward 4-methykatechol was 60.6% of that of catechol, while the activity toward 3-methylcatechol and 4-chlorocatechol were 27.0 and 13.5%, respectively.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.763-771
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• 2001

Biphenyl dioxygenase (BPDO), which catalyzes the first step in the bacterial degradation of biphenyl and polychlorinated biphenyls, was characterized in Pseudomonas sp. B4. The bphA locus containing the four structural genes encoding BPDO were cloned and sequenced. A regulatory gene as well as a putative regulatory sequence were identified upstream of this locus. A transposase-like gene was found within a 1-kb region further upstream, thereby suggesting that the bphA locus may be carried on a transposable element. The three components of the BPDO enzyme have been separately overexpressed and purified from E. coli. The ferredoxin and terminal dioxygenase components showed biochemical properties comparable to those of two previously characterized BPDOs, whereas the ferredoxin reductase exhibited an unusually high lability. The substrate selectivity of BPDO was examined in vivo using resting cell assays performed with mixtures of selected polychlorinated biphenyls. The results indicated that para-substituted congeners were the preferred substrates. In vitro studies were carried out on a BPDO complex where the reductase from strain B4 we replaced by the more stable isoform from Comamonas testosteroni B-356. The BPDO enzyme had a specific activity of $0.26{\pm}0.02 {\mu}mol {min^-1}{mg^-1}\;of\;ISP_{BPH}$ with biphenyl as the substrate. The 2,3-, 4,4'-, and 2,4,4'-chlorobiphenyls were converted to single dihydrodiols, while 2,4'-dichlorobiphenyl gave rise to two dihydrodiols. The current data also indicated that 2,4,4'-trichlorobiphenyl was a better substrate than the 4,4'-dichlorinated congener.

• Journal of Applied Biological Chemistry
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• v.43 no.1
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• pp.44-48
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• 2000

Burkholderia cepacia strain G4 (pHG-2) containing toluene 2-monooxygenase and toluene dioxygenase, was able to grow on toluene and accumulate cis-3-methyl-3,5-cyclohexadien-1,2-diol (cis-toluene dihydrodiol) in the liquid culture. The cis-toluene dihydrodiol produced was identical to the authentic compound, as judged through mass spectrometry and nuclear magnetic resonance analysis. Our results indicate that pHG-2 provides an economical means to produce chemically-important chiral synthons while growing on toluene.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1440-1445
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• 2010

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 were 8.0, $30^{\circ}C$, and 175 rpm, respectively. In addition, low concentrations of glucose were found to inhibit the degradation of DBP, whereas high concentrations of glucose increased its degradation. Meanwhile, a substrate utilization test showed that JDC-11 was also able to utilize other phthalates. The major metabolites of DBP degradation were identified as monobutyl phthalate and phthalic acid by gas chromatography-mass spectrometry, allowing speculation on the tentative metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11. Using a set of new degenerate primers, a partial sequence of the 3,4-phthalate dioxygenase gene was obtained from JDC-11. Moreover, a sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of the phthalate dioxygenase from Rhodococcus coprophilus strain G9.

• BMB Reports
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• v.35 no.4
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• pp.432-436
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• 2002

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.487-494
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• 2003

2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO), an enzyme of the biphenyl biodegradation pathway encoded by the bphC gene of Comnmonas sp. SMN4, was expressed and purified using column chromatographies. SDS-PAGE of purified 23DBDO showed a single band with a molecular mass of 32 kDa, which was consistent with the data from the gel filtration chromatography (GFC). The purified enzyme exhibited a maximum 23DBDO activity at pH 9.0 and was stable at pH 8.0. The enzyme showed maximum activity at $40^{\circ}C$ and maintained activity at $30^{\circ}C$ for 24 h. Kinetic parameters represented by Michaelis-Menten constants such as $K_m\;and\;V_{max}$ values for various substrates were determined by Lineweaver-Burk plots: The purified enzyme 23DBDO from Comamonas sp. SMN4 had the highest catalytic activity for 2,3-dihydroxybiphenyl and 3-methylcatechol, and had very poor activity with catechol and 4-methylcatechol.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.264-269
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• 1998

Commamonas acidovorans SMN4 showed wide growth substrate spectra for various aromatic hydrocarbons. Strain SMN4 was able to grow on biphenyl producing a meta-cleavage compound, yellow 2-hydroxy-6-oxophenylhexa-2,4-dienoic acid with a spray of 2,3-dihydroxybiphenyl, while it also grew on catechol, developing yellow 2- hydroxymucoic semialdehyde with a spray of 100 mM catechol. Thus these results indicate that two-ring structures of biphenyl were cleaved by meta-mode in upper and lower pathways. Strain SMN4 metabolized various substituted biphenyl compounds and xylene to the corresponding benzoate derivatives through oxidation of the ring structures. It was clearly shown that biphenyl can be a common inducer in the oxidation of biphenyl and 2,3-dihydroxybiphenyl. Various compounds were examined for their suitability to serve as substrates for indole oxidation, indicating that biphenyl, benzoate, and succinate are quite good inducers of indigo production due to the activity of biphenyl dioxygenase. This results suggest that indigo formation is by means of the combined activities of biphenyl dioxygenase and tryptophanase.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.416-421
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• 1998

A carboxyl group modifier, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to study the interactions between three components of toluene dioxygenase (TDO) from Pseudomonas putida FI. $Ferredoxin_{TOL}$ activity was increased by the treatment with EDC; however, the activity was rapidly declined in the prolonged incubation. In covalent cross-linking experiments with EDC, $Ferredoxin_{TOL}$ made a one-to-one complex with $Reductase_{TOL}$ or the large subunit of $ISP_{TOL}$. These results provide evidence for the involvement of electrostatic interactions in the TDO electron transfer system.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.352-357
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• 1998

Biodegradation of benzoate and m-toluate was investigated using a Pseudomonas sp. isolated in a continuous culture for 45 days with a step-wise increase of the subsrates. The optimum mixture ratio of benzoate and m-toluate was 75% and 25%, respectively. During 45-day culture, removal of benzoate and m-toluate, which was replaced 2,000 ppm on the 30th day were 94% and 79%, respectively, when COD removal rate was 80%. The enzymatic activity of catechol 1,2-dioxygenase increased and that of catechol 2,3-dioxygenase decreased as the concentration of m-toluate was increased. These results suggested that m-toluate induced enzyme activity for degradation of benzoate. The shape of isolated strain in the continuous culture was investigated with SEM and the results showed that the cell shape was more damage according to the higher concentration of aromatic hydrocarbons. Therefore, we suggested that the tolerance against aromatic hydrocarbons was related to not only enzymatic activity but also characteristic of cell membrane or cell wall.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.249-254
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• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.