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1.
We compared the metabolism of eight di- and trichlorobiphenyls by eight bacterial strains chosen to represent a broad range
of degradative activity against polychlorinated biphenyls (PCBs). The PCB congeners used were 2,3-, 2,3′-, 2,4′-, 3,3′-, 2,3,3′-,
2,4,4′-, 2,5,3′-, and 3,4,2′-chlorobiphenyl. The bacterial strains used wereCorynebacterium sp. MB1,Alcaligenes strainsA. eutrophus H850 andA. faecalis Pi434, andPseudomonas strains LB400 and H1130,P. testosteroni H430 and H336, andP. cepacia H201. The results indicated that both the relative rates of primary degradation of PCBs and the choice of the ring attacked
were dependent on the bacterial strain used. The bacterial strains exhibited considerable differences in their relative reactivity
preferences for attack on mono- and dichlorophenyl groups and in the degree to which the attack was affected by the chlorine
substitution pattern on the nonreacting ring. For MB1 the reactivity pattern was 3-≥4-≫2-chlorophenyl with no attack on 2,4-
or 2,5-chlorophenyl groups. This strain was relatively insensitive to the chlorine substitution pattern on the nonreacting
ring. Strains H1130, H430, H201, and Pi434 exhibited the same reactivity preferences as MB1, but for these strains (and for
all others tested) the chlorination pattern on the nonreacting ring had a strong effect. For strain H336 the reactivity preference
was 4-≥2->2,4-≥3-chlorophenyl, with no evidence of attack on 2,5-chlorophenyl rings. For strains H850 and LB400 the relative
reactivity was 2->2,5->3-≫2,4->4-chlorophenyl. On this basis we propose that the eight bacterial strains represent four distinct
classes of biphenyl/PCB-dioxygenase activity.
The types of products formed were largely strain-independent and were determined primarily by the chlorine substitution pattern
on the reacting ring. When the reacting ring was an unsubstituted phenyl or a 2-chlorophenyl group, the products were chlorobenzoic
acids in high yields; for a 3-chlorophenyl ring, both chlorobenzoic acids and chloroacetophenones in moderate yields; and
for a 4- or 2,4-chlorophenyl group, chlorobenzoic acids in low yields with an apparent accumulation ofmeta ring-fission product. Strains H850 and LB400 were able to degrade the 3-chlorobenzoic acid that they produced from the degradation
of 2,3′-chlorobiphenyl. We conclude that despite differences among strains in the specificity of the initial dioxygenase,
the specificities of the enzymes responsible for the subsequent degradation to chlorobenzoic acid and/or chloroacetophenone
are quite similar for all strains. 相似文献
2.
Growth on dichlorobiphenyls with chlorine substitution on each ring by bacteria isolated from contaminated African soils 总被引:4,自引:0,他引:4
Adebusoye SA Picardal FW Ilori MO Amund OO Fuqua C Grindle N 《Applied microbiology and biotechnology》2007,74(2):484-492
Until recently, it was generally believed that the presence of more than one chlorine substituent prevented chlorinated biphenyls
from serving as a sole source of carbon and energy for aerobic bacteria. In this study, we report the isolation of three aerobic
strains, identified as Enterobacter sp. SA-2, Ralstonia sp. SA-4, and Pseudomonas sp. SA-6 from Nigerian polluted soils, that were able to grow on a wide range of dichlorobiphenyls (diCBs). In addition to
growing on all monochlorobiphenyls (monoCBs), the strains were all able to utilize 2,2′-, 2,4′-, and 2,3-diCB as a sole source
of carbon and energy. With the exception of strain SA-2, growth was also sustainable on 3,3′-, and 3,5-diCB. Washed benzoate-grown
cells were typically able to degrade 68 to 100% of the diCB (100 ppm) within 188 h, concomitant with a cell number increase
of up to three orders-of-magnitude and elimination of varying amounts of chloride. In many cases, stoichiometric production
of a chlorobenzoate (CBA) as a product was observed. During growth on 2,2′-, and 2,4′-diCB, organisms exclusively attacked
an o-chlorinated ring resulting in the production of 2-CBA and 4-CBA, respectively. A gradual decline in the concentration of
the latter was observed, which suggested that the product was being degraded further. In the case of 2,3-diCB, the unsubstituted
ring was preferentially metabolized. Initial diCB degradation rates were greatest for 2,4′-diCB (11.2 ± 0.91 to 30.3 ± 7.8 nmol/min
per 109 cells) and lowest for 2,2′-diCB (0.37 ± 0.12 to 2.7 ± 1.2 nmol/min per 109 cells). 相似文献
3.
K. A. Billingsley S. M. Backus O. P. Ward 《Applied microbiology and biotechnology》1999,52(2):255-260
A variety of commercial surfactants were tested to determine their effect on polychlorinated biphenyl (PCB) transformation
by Pseudomonas LB400. Initial tests determined that most surfactants were fully or partially able to solubilize the PCB congeners 2,5,2′-chlorobiphenyl
(CBP), 2,4,2′,4′-CBP, 2,3,5,2′,5′-CBP and 2,4,5,2′,4′,5′-CBP, at concentrations above the surfactants' critical micelle concentration
(CMC). Surfactants were also found to have no negative effect on bacterial survival, as cell numbers were the same or higher
after incubation in the presence of surfactants than after incubation without surfactants. A comparison of the extent of biotransformation
of single PCB congeners by the bacterium revealed that, at surfactant concentrations above the CMC, the presence of an anionic
surfactant promoted while nonionic surfactants inhibited PCB transformation, compared to a control with no surfactant. The
rates of transformation of PCB congeners were also higher in the presence of the anionic surfactant compared to the control.
The inhibitory effects of a nonionic surfactant, Igepal CO-630 at a concentration above its CMC, on transformation of 2,4,5,2′,5′-CBP
could be eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant CMC.
Received: 26 October 1998 / Received revision: 5 March 1999 / Accepted: 14 March 1999 相似文献
4.
Juan Matías Saavedra Francisca Acevedo Myriam González Michael Seeger 《Applied microbiology and biotechnology》2010,87(4):1543-1554
Polychlorobiphenyls (PCBs) are classified as “high-priority pollutants.” Diverse microorganisms are able to degrade PCBs.
However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end
metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading
bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically
modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA,
and degraded 3,5-CBA in the presence of m-toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4′-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted
soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of
2,4′-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost
two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation
of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the
development of improved biocatalysts for remediation of PCB-contaminated environments. 相似文献
5.
Pseudomonas sp. D7-4 and Pseudomonas sp. B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chlorobenzoate alone. The competition behaviour
of these strains was compared in batch and in chemostat cultures. Despite misrouting of metabolites, strain WR201 was competitive,
in a lot of the competition experiments, with mixtures of these substrates. Only in a narrow range of the mixing ratio of
chloro- and methylbenzoate was the presence of both the meta and ortho pathways a disadvantage for competitiveness. Outside these ranges other attributes, such as high growth rates or short lag
periods, of a respective strain were even more essential for one strain to outcompete another.
Received: 13 February 1998 / Received revision: 28 April 1998 / Accepted: 30 April 1998 相似文献
6.
Microbial Growth on Dichlorobiphenyls Chlorinated on Both Rings as a Sole Carbon and Energy Source 总被引:5,自引:0,他引:5 下载免费PDF全文
We have isolated bacterial strains capable of aerobic growth on ortho-substituted dichlorobiphenyls as sole carbon and energy sources. During growth on 2,2′-dichlorobiphenyl and 2,4′-dichlorobiphenyl strain SK-4 produced stoichiometric amounts of 2-chlorobenzoate and 4-chlorobenzoate, respectively. Chlorobenzoates were not produced when strain SK-3 was grown on 2,4′-dichlorobiphenyl. 相似文献
7.
M. H. A. van Eekert A. J. M. Stams J. A. Field G. Schraa 《Applied microbiology and biotechnology》1999,51(1):46-52
The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor
was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product
formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted,
although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors
like vitamin B12 and F430. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although
at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation
of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with
the Gibbs free-energy change (ΔG
0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have
a higher ΔG
0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the
methanogenic granular sludge.
Received: 8 June 1998 / Received revision: 7 September 1998 / Accepted: 13 September 1998 相似文献
8.
Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 of Pseudomonas pseudoalcaligenes KF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes in Escherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2′-dichlorobiphenyl and 2,5,4′-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2′-dichlorobiphenyl, forming 2-chloro-2′,3′-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4′-trichlorobiphenyl via both 2′,3′- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox of Burkholderia xenovorans LB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.Electronic supplementary material Supplementary material is available in the online version of this article at . 相似文献
9.
Matthew O. Ilori Gary K. Robinson Sunday A. Adebusoye 《World journal of microbiology & biotechnology》2008,24(8):1259-1265
Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor
1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4′-dichlorobiphenyl (4,4′-diCB)
and a wide range of other xenobiotic compounds. During growth on 4,4′-diCB, a near-stoichiometric amount of chloride was excreted
into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production
of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4′-diCB and
biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4′-diCB was mineralized
in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM
CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth
was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important
advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil. 相似文献
10.
A biphenyl-utilizing bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soils grew on tryptic soy at temperatures
between 4 and 40°C. The Gram-negative rod bacterium formed yellow colonies on nutrient agar and it denitrified nitrate to
nitrogen. Analysis of cellular fatty acids showed that it was most closely related to Hydrogenophaga taeniospiralis. At 5°C, biphenyl-grown cells cometabolically degraded di- and trichlorinated isomers of PCBs in 10 ppm of Aroclor 1248.
At 30°C, PCBs that were removed included a congener with four chlorine substituents. At 5°C, cells transformed 2,4′-dichlorobiphenyl
(2,4′-DCB) and accumulated ortho-chlorinated meta-cleavage product as a stable metabolite. Analysis of extracts of culture supernatant by gas chromatography–mass spectrometry
indicated that products of transformation of 2,4′-DCB included 2- and 4-chlorobenzoic acid (2- and 4-CBA), suggesting that
(chloro)biphenyl-degrading upper-pathway enzymes of the bacterium are active at low temperature. The bacterium Hydrogenophaga sp. IA3-A is a PCB-degrading psychrotolerant strain. 相似文献
11.
Thi Thanh My Pham Mohammad Sondossi Michel Sylvestre 《Applied and environmental microbiology》2015,81(14):4860-4872
In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them. 相似文献
12.
S. A. Kozlovsky G. M. Zaitsev F. Kunc J. Gabriel A. M. Boronin 《Folia microbiologica》1993,38(5):371-375
A strain ofPseudomonas stutzeri KS25 utilizing 2-chlorobenzoic and 2,5-dichlorobenzoic acids as the sole carbon and energy source was isolated from polychlorophenol-contaminated
soil and sewage, using the method of enrichment cultures. This strain was also able to grow on 2-fluoro-, 2-iodo-, 2-bromo-
and 2,5-dihydroxybenzoate, but did not utilize 3-, 4-chloro-, 2,4- and 2,6-dichlorobenzoates as the sole carbon and energy
source, however, it cometabolized 3-chloro-, 2,4-and 2,6-dichlorobenzoates, but not 4-chlorobenzoate. The yield of released
chlorine during utilization of 2-chloro- and 2,5-dichlorobenzoates amounted to 100 % of the theoretical. The concentration
of 2-chloro- and 2,5-dichlorobenzoates, not substantially inhibiting the isolated microorganism, was within the range 0.25–0.5
and 2.5–3.0 g/L, respectively. 相似文献
13.
G. Mukerjee-Dhar Takashi Hatta Minoru Shimura Kazuhide Kimbara 《Archives of microbiology》1997,169(1):61-70
We isolated and characterized a gram-negative bacterium, Burkholderia sp. strain TSN101, that can degrade polychlorinated biphenyls (PCBs) at concentrations as high as 150 μg Kaneclor 300/ml,
a PCB mixture equivalent to Aroclor 1242. Growing cells of strain TSN101 degraded most of the tri- and tetrachlorobiphenyls
in medium containing 25 μg Kaneclor 300/ml. Using PCB concentrations of 50–150 μg of Kaneclor 300/ml, the congener selectivity
pattern was different and the pattern of chlorine substitution strongly affected degradation of some congeners. At 25 μg Kaneclor
300/ml, strain TSN101 degraded di- and trichlorinated congeners with chlorine substitutions at both the ortho and the para positions. At higher concentrations of Kaneclor 300, di- and trichlorobiphenyls with ortho substituents in both phenyl rings were not degraded well. Trichlorobiphenyls with para and meta substitutents were degraded equally well at all concentrations studied. The ability of strain TSN101 to degrade ortho and para-substituted congeners was confirmed using a defined PCB mixture with chlorine substituents at 2′- and 4′-positions. A 5-kb
DNA fragment containing the bphBCD genes was cloned and sequenced. Comparison of the deduced amino acid sequences of these genes with related proteins indicated
99 and 98% sequence similarity to the BphB and BphD of Comamonas testosteroni strain B-356, respectively. The bphC gene product showed 74% sequence similarity to the BphC of Burkholderia cepacia strain LB400 and exhibited a narrow substrate specificity with strong affinity for 2,3-dihydroxybiphenyl. A bphC-disrupted mutant of Burkholderia sp. strain TSN101, constructed by gene replacement, lost the ability to utilize biphenyl, thus supporting the role of the
cloned bph gene in biphenyl metabolism.
Received: 18 February 1997 / Accepted: 19 August 1997 相似文献
14.
Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased
final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent
productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch
was necessary, indicating high activity of the clostridial amylases produced by the strain applied.
Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998 相似文献
15.
G. H. van Geel-Schutten F. Flesch B. ten Brink M. R. Smith L. Dijkhuizen 《Applied microbiology and biotechnology》1998,50(6):697-703
A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with
high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS.
Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in
the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose)
supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This
is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes.
Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998 相似文献
16.
A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from
activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and
84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly 14C-labelled DNOC was recovered as 14CO2. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol,
2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads
in fixed-bed column reactors were 30 mmol day−1 (l reactor volume)−1, leaving an effluent concentration of less than 5 μg l−1 DNOC in the outflowing medium. The apparent K
s value of the immobilized mixed culture for DNOC was 17 μM. Degradation was inhibited at DNOC concentrations above 30 μM and
it ceased at 340 μM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing
mechanism.
Received: 27 March 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997 相似文献
17.
Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls 下载免费PDF全文
Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISPBPH of strain LB400 and the β subunit of ISPBPH of strain B-356 (the αLB400βB-356 chimera) and the αB-356βLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the αB-356βLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the αLB400βB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl. 相似文献
18.
We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet
radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of
DNA-damaging agents in the environment.
Received: 18 February 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998 相似文献
19.
M. D. Vollmer U. Schell V. Seibert S. Lakner M. Schlömann 《Applied microbiology and biotechnology》1999,51(5):598-605
The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted
cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different
from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k
cat/K
m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes.
TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred
substrate.
Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998 相似文献
20.
Martina McGuinness Claire Ivory Niamh Gilmartin David N. Dowling 《International biodeterioration & biodegradation》2006,58(3-4):203
The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphKLB400, with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphKLB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphKLB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphKLB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphKLB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphKLB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation. 相似文献