• Journal of Microbiology
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• v.35 no.2
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• pp.141-148
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• 1997

Alcaligenes xylosoxydans strain SMN3 capable of utilizing biphenyl grew not only on phenol, and benzoate, but also on salicylate. Catabolisms of biphenyl and salicylate appear to be interrelated since benzoate is a common metabolic intermediate of these compounds. Enzyme levels in the excatechol 2. 3-dioxygenas which is meta-cleavage enzyme of catechol, but did not induce catechol 1, 2-dioxygenase. All the oxidative enzymes of biphenyl and 2, 3,-dihydroxybiphenyl (23DHBP) were induced when the cells were grown on biphenyl and salicylate, respectively. Biphenyl and salicylate could be a good inducer in the oxidation of biphenyl and 2, 3-dihydroxybiphenyl. The two enzymes for the degradation of biphenyl and salicylate were induced after growth on either biphenyl or salicylate, suggesting the presence of a common regulatory element. However, benzoate could not induce the enzymes responsible for the oxidation of these compounds. Biphenyl and salicylate were good inducers for indigo formation due to the activity of biphenyl dioxygenase. These results suggested that indole oxidation is a property of bacterial dioxygenase that form cis-dihydrodiols from aromatic hydrocarbon including biphenyl.

• 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
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• v.40 no.1
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• pp.86-89
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• 2002

Rhodococcus sp. strain T104, which is able to grow on either biphenyl or limonene, was found to utilize phenol as sole carbon and energy sources. Furthermore, T104 was positively identified to possess three separate pathways for the degradation of limonene, phenol, and biphenyl. The fact that biphenyl and limonene induced almost the same amount of catechol 1,2-dioxygenase activity indicates that limonene can induce both upper and lower pathways for biphenyl degradation by T104.

• Microbiology and Biotechnology Letters
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• v.24 no.6
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• pp.657-663
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• 1996

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

• 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.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.48-51
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• 1992

Achromobacter xylosoxidans KF701 and Pseudomonas putida (NAH7) were significantly different in degradative capability of aromatic compounds including benzoates, biphenyls, and naphthalene. However, both of the bacterial strains can grown on catechol as the sole carbon and energy source. Catechol 2, 3-dioxygenase gene for naphthalene oxidation or biphenyl oxidation was cloned into Escherichia coli HB 701. A E. coli HB 101 clone containing catechol 2, 3-dioxygenase gene from P. putida (NAH7) contains a recombinant plasmid with 3.60kb pBR322 and 6-kb insert DNA. Another E. coli HB101 clone containing catechol 2, 3-dioxygenase gene from A. xylosoxidans KF 701 has a recombinant plasmid with 4.4kb pBR322 and 10-kb insert DNA. Physical maps of the recombinant plasmids were constructed, and catechol 2, 3-dioxygenase gene in the recombinant plasmide was further localized and subcloned int M13. The cloned-catechol 2, 3-dioxygenase game products were identified as yellow bands on nondenaturaing polyacrylamide gel after electrophoresis followed by activity staining with catechol solution.

• 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.

• Proceedings of the Microbiological Society of Korea Conference
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• 2001.11a
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• pp.16-25
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• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

• Proceedings of the PSK Conference
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• 2001.04a
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• pp.173.2-173.2
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• 2001

• Microbiology and Biotechnology Letters
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• v.21 no.2
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• pp.150-156
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• 1993

The 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase, the product of pcbC gene, was purified from the biphenyl and 4-chlorobiphenyl degrading Pseudomonas sp. DJ-12 by the methods of acetone precipitation, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-150 gel filtration chromatography. The enzyme was estimated to be about 260 kilodaltons in molecular weight and to be consisted of eight subunits. The Km value of the enzyme was 61 nM to 2,3-DHBP and the highest activity of the enzyme was observed at pH 8 and 30C.