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PubMed1992

Modelling of mixed chemostat cultures of an aerobic bacterium, Comamonas testosteroni, and an anaerobic bacterium, Veillonella alcalescens: comparison with experimental data.

A mathematical model of mixed chemostat cultures of the obligately aerobic bacterium Comamonas testosteroni and the anaerobic bacterium Veillonella alcalescens grown under dual limitation of L-lactate and oxygen was constructed. The model was based on Michaelis-Menten-type kinetics for the consumption of substrates, with noncompetitive inhibition of V. alcalescens by O2. The growth characteristics of the aerobic and anaerobic organisms were determined experimentally with pure cultures of the individual species in (oxygen-limited) chemostats. Using these pure-culture data in the model of the mixed culture resulted in a good description of the actual mixed cultures of the two bacteria. In the actual mixed-culture experiments, coexistence of the two species occurred only when the cultures were oxygen limited. With increasing oxygen supply (the actual oxygen concentration in the culture remaining at less than 0.2 microM), the biomass of C. testosteroni increased, whereas that of V. alcalescens decreased. Apparently, C. testosteroni protected V. alcalescens from inhibition by oxygen by maintaining sufficiently low oxygen concentrations. The model calculations indicated that competition between the aerobic and the anaerobic bacterium for common substrates (L-lactate and oxygen) occurred and that the anaerobe was the better competitor. Analysis of the culture fluid indicated that C. testosteroni grew primarily at the expense of the fermentation products of V. alcalescens, i.e., propionate and acetate. The model further indicated that with different values of several growth parameters (e.g., substrate affinity and/or inhibition constants), the affinity of the aerobic organism for oxygen and the sensitivity of the anaerobic organism for oxygen were the most important properties determining the coexistence of these two physiologically different types of bacteria.

Acetates↗

PubMed1995

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

Amino Acid Sequence↗

PubMed1996

Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases.

In a previous work, all three components of Comamonas testosteroni B-356 biphenyl (BPH)/chlorobiphenyls (PCBs) dioxygenase (dox) have been purified and characterized. They include an iron-sulphur protein (ISPBPH) which is the terminal oxygenase composed of two subunits (encoded by bphA and bphE), a ferredoxin (FERBPH) encoded by bphF and a reductase (REDBPH) encoded by bphG. bphG Is not located in the neighbourhood of bphAEF in B-356. We are reporting the cloning of B-356-bphG and the sequencing of B-356-BPH dox genes. Comparative analysis of the genes provided genetic evidence showing that two BPH dox lineages have emerged in Gram-negative bacteria. The main features of the lineage that includes B-356 are the location of bphG outside the bph gene cluster and the structure of REDBPH which is very distinct from all other aryl dioxygenase-reductases.

Amino Acid Sequence↗

PubMed1995

Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.

Delta 5-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catalyzes the isomerization of a wide variety of delta 5(6) and delta 5(10) steroids through the formation of an enzyme bound dienol(ate) intermediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed kinetic characterization of the KSI mutants D38E and D38H are presented. Both mutants retain significant activity, with kcat and kcat/Km values 10(3)-10(4) times greater than the D38N mutant. The results allow for a qualitative assessment of the sensitivity of the enzymes catalytic capability to the positioning and chemical nature of the catalytic base. The near identity of the ratios of kcat5-AND/kcat5,10-EST is most easily explained by a mechanism in which the second chemical step, reketonization of the intermediate dienol(ate), is not significantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier proposals that an as yet unidentified titrating functional group is present in the active site and indicates that the electrostatic environment of residue 38 is hydrophobic and positively charged.

Androstenedione↗

PubMed1994

Description of the kinetic mechanism and the enantioselectivity of quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni in the oxidation of alcohols and aldehydes.

Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa-uni ping-pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)- and (S)-solketal [2,2-dimethyl-4-(hydroxymethyl)-1,3-dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.

1-Propanol↗

PubMed1995

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Alcohol Oxidoreductases↗

PubMed1995

Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity.

The nucleotide sequence of bphD, encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway of Comamonas testosteroni strain B-356, was determined. Comparison of the deduced amino-acid sequence with published sequences led to the identification of a 'lipase box', containing a consensus pentapeptide sequence GlyXaaSerXaaGly. This suggested that the mechanism of action of this enzyme may involve an Asp-Ser-His catalytic triad similar to that of classical lipases and serine hydrolases. Further biochemical and genetic evidence for the active-site involvement of Ser112 was obtained by showing that a semipurified enzyme was inhibited by PMSF, a classic inhibitor of serine hydrolases, and by site-directed Ser112-->Ala mutagenesis.

Amino Acid Sequence↗

PubMed1996

Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB).

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Amino Acid Sequence↗

PubMed1993

Microbial metabolism of quinoline and related compounds. XVII. Degradation of 3-methylquinoline by Comamonas testosteroni 63.

A bacterial strain which utilizes 3-methylquinoline as sole source of carbon, nitrogen and energy was isolated from activated sludge. On the basis of its morphological and physiological characteristics, this isolate was classified as Comamonas testosteroni. Four metabolites of 3-methylquinoline degradation were isolated from the culture supernatant and identified as 3-methyl-2-oxo-1,2-dihydroquinoline, 6-hydroxy-3-methyl-2-oxo-1,2-dihydroquinoline, 5,6-dihydroxy-3-methyl-2-oxo-1,2-dihydroquinoline and 2,5,6-trihydroxy-3-methylpyridine. Based on these results, a degradation pathway for 3-methylquinoline is proposed.

Culture Media↗

PubMed1996

Degradative pathways for p-toluenecarboxylate and p-toluenesulfonate and their multicomponent oxygenases in Comamonas testosteroni strains PSB-4 and T-2.

Three multicomponent oxygenases involved in the degradation of p-toluenesulfonate and p-toluenecarboxylate and the regulation of their synthesis have been examined in three strains (T-2, PSB-4 and TER-1) of Comamonas testosteroni. Strain T-2 utilizes p-toluenesulfonate as a source of carbon and energy for growth via p-sulfobenzoate and protocatechuate, and p-toluenecarboxylate via terephthalate and protocatechuate, and has the unusual property of requiring the reductase (TsaB) of the toluenesulfonate methyl monooxygenase system (TsaMB) in an incompletely expressed sulfobenzoate dioxygenase system (PsbAC) [Schläfli Oppenberg, H.R., Chen, G., Leisinger, T. & Cook, A. M. (1995). Microbiology 141, 1891-1899]. The independently isolated C. testosteroni PSB-4 utilized only sulfobenzoate and terephthalate via protocatechuate. Mutant TER-1, derived from strain T-2, utilized only terephthalate via protocatechuate. We detected no enzymes of the pathway from toluenesulfonate to sulfobenzoate in strains PSB-4 and TER-1, and confirmed by PCR and Southern blot analysis that the genes (tsaMB) encoding toluenesulfonate monooxygenase were absent. We concluded that, in strain PSB-4, the regulatory unit encoding the genes for the conversion of toluenesulfonate to sulfobenzoate was missing, and that generation of mutant TER-1 involved deletion of this regulatory unit and of the regulatory unit encoding desulfonation of sulfobenzoate. The degradation of sulfobenzoate in strain PSB-4 was catalysed by a fully inducible sulfobenzoate dioxygenase system (PsbACPSB-4), which, after purification of the oxygenase component (PsbAPSB-4), turned out to be indistinguishable from the corresponding component from strain T-2 (PsbAT-2). Reductase PsbCPSB-4, which we could separate but not purify, was active with oxygenase PsbAPSB-4 and PsbAT-2. Oxygenase PsbAPSB-4 was shown by electron paramagnetic resonance spectroscopy to contain a Rieske [2Fe-2S] centre. The enzyme system oxygenating terephthalate was examined and the oxygenase component purified and characterized. The oxygenase component in strains T-2 (and mutant TER-1) and PSB-4 were indistinguishable. The reductase component, which we separated but failed to purify, was active with the oxygenase from all strains. Gains and losses of blocks of genes in evolution is discussed.

Benzenesulfonates↗

PubMed2001

Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer.

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution. It comprises two domains. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.

Alcohol Oxidoreductases↗

PubMed2003

Deuterium isotope effect on enantioselectivity in the Comamonas testosteroni quinohemoprotein alcohol dehydrogenase-catalyzed kinetic resolution of rac-2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, solketal.

Isotopic substitution provides an effective tool to probe the mechanism of enzyme-catalyzed reactions. To our knowledge, kinetic isotope effects on the enantioselectivity of enzymes have not been reported. We investigated the effect of deuterium substitution on the enantiomeric ratio, E, of PQQ-containing quinohemoprotein alcohol dehydrogenase, QH-ADH, from Comamonas testosteroni in the ferricyanide-coupled kinetic resolution of rac-2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, solketal. Under otherwise identical conditions, we measured E=30 for solketal and E=6 for rac-2,2-dimethyl-4-[1,1-2H]hydroxymethyl-1,3-[5,5,4-2H]dioxolane, d(5)-solketal. It is proposed that isotopic substitution affects the relative kinetic weights of the initial hydron/deuteron transfer from substrate to cofactor and the subsequent proton/deuteron shift in the cofactor-product complex. The latter step becomes more important in the deuterated complex to the extent that the enantiomer discrimination in the first step is partially overruled.

Alcohol Oxidoreductases↗

PubMed1995

Characterization by arbitrary primer polymerase chain reaction of polychlorinated biphenyl (PCB)-degrading strains of Comamonas testosteroni isolated from PCB-contaminated soil.

In this study, we isolated and characterized biphenyl (BP) and polychlorinated biphenyl (PCB) degrading bacterial strains found in PCB-contaminated soil from an auto manufacturing plant located in Syracuse, New York. Twenty-one BP and PCB-degrading bacteria were randomly selected to form a representative sample of the bacterial population present at the site. Of the 21 bacteria, 13 were identified as Comamonas testosteroni, constituting about 60% of the bacterial population examined. Other PCB degraders identified were Acidovorax facilis, Alcaligenes xylosoxydans, Bacillus sphericus, Hydrogenophaga pseudoflava, Pseudomonas avanae, and Rhodococcus fascians. Owing to the abundance of C. testosteroni at this site, only these isolates were further characterized for their PCB congener degradation profile, 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and genetic relatedness by polymerase chain reaction (PCR) analysis. The PCB congener degradation pattern revealed a high degree of variability among the C. testosteroni isolates. The majority of the C. testosteroni isolates tested could degrade more than 95% of the PCB congeners up to pentachlorinated biphenyl. Only four isolates could degrade more than 80% of hexachlorobiphenyl. All 12 isolates of C. testosteroni tested were able to attack 2,3,4,5,6,3',4'-heptachlorobiphenyl, indicating involvement of biphenyl 2,3-dioxygenase, while 2,3,5,6,2',3',6'-heptachlorobiphenyl was attacked by 6 strains, suggesting an oxidation reaction mediated by 3,4-dioxygenase. 2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was also found to vary among the C. testosteroni isolates tested in this study. Eleven strains showed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity specific for 2,3-dihydroxybiphenyl, whereas isolate BW 169 could metabolize both 2,3-dihydroxybiphenyl and 4-methylcatechol, and isolate BW74 had the ability to metabolize all three substrates (2,3-dihydroxybiphenyl, 4-chlorocatechol, and 4-methylcatechol).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed2006

Treatment of naphthalene-2-sulfonic acid from tannery wastewater by a granular activated carbon fixed bed inoculated with bacterial isolates Arthrobacter globiformis and Comamonas testosteroni.

The kinetics of naphthalene-2-sulfonic acid (2-NSA) adsorption by granular activated carbon (GAC) were measured and the relationships between adsorption, desorption, bioavailability and biodegradation assessed. The conventional Langmuir model fitted the experimental sorption isotherm data and introduced 2-NSA degrading bacteria, established on the surface of the GAC, did not interfere with adsorption. The potential value of GAC as a microbial support in the aerobic degradation of 2-NSA by Arthrobacter globiformis and Comamonas testosteroni was investigated. Using both virgin and microbially colonised GAC, adsorption removed 2-NSA from the liquid phase up to its saturation capacity of 140 mg/g GAC within 48 h. However, between 83.2% and 93.3% of the adsorbed 2-NSA was bioavailable to both bacterial species as a source of carbon for growth. In comparison to the non-inoculated GAC, the combination of rapid adsorption and biodegradation increased the amount (by 70-93%) of 2-NSA removal from the influent phase as well as the bed-life of the GAC (from 40 to >120 d). A microbially conditioned GAC fixed-bed reactor containing 15 g GAC removed 100% 2-NSA (100 mg/l) from tannery wastewater at an empty bed contact time of 22 min for a minimum of 120 d without the need for GAC reconditioning or replacement. This suggests that small volume GAC bioreactors could be used for tannery wastewater recycling.

Adsorption↗

PubMedDate not supplied

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni.

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences.

Biodegradation, Environmental↗

PubMed2003

Gene encoding the hydrolase for the product of the meta-cleavage reaction in testosterone degradation by Comamonas testosteroni.

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.

Bacterial Proteins↗

PubMed2005

Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha.

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthalenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25 degrees C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2-n11). The loss of individual oligomers was correlated with the length of the NSA-CH2 chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2-n11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25 degrees C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6-n11 were degraded after 120 h. At a higher temperature (37 degrees C) oligomers n4-n11 were degraded completely after 120 h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5 g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C. polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.

Adsorption↗

PubMed2001

Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni B-356.

The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 alpha subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO alpha subunit with that of LB400 alpha subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.

Burkholderia↗