04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Enrichment of dibenzofuran utilizing bacteria with high co-metabolic potential towards dibenzodioxin and other anellated aromatics(1989) Strubel, Volker; Rast, Hans G.; Fietz, Walter H.; Knackmuss, Hans-Joachim; Engesser, Karl-HeinrichDibenzofuran degrading bacteria were enriched from various environmental sources. A mutualistic mixed culture of strain DPO 220 and strain DPO 230 was characterized. Strain DPO 220 alone showed limited growth with dibenzofuran as sole source of carbon and energy (td ≥ 4.5 h). A labile degradation product, C12H10O5, and salicylate were isolated from the culture fluid. Salicylate was found to be a central intermediate of DBF-degradation.Strain DPO 220 co-metabolized a wide range of anellated aromatics as well as heteroaromatics. High rates of co-oxidation of dibenzodioxin demonstrate analogue-enrichment to be a powerful technique for selecting enzymatic activities for otherwise non-degradable substrates.Item Open Access (+)-4-Carboxymethyl-2,4-dimethylbut-2-en-4-olide as dead-end metabolite of 2,4-dimethylphenoxyacetic acid or 2,4-dimethylphenol by alcaligenes eutrophus JMP 134(1990) Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim2,4-Dimethylphenoxyacetic acid and 2,4-dimethylphenol are not growth substrates for Alcaligenes eutrophus JMP 134 although being cooxidized by 2,4-dichlorophenoxyacetate grown cells. None of the relevant catabolic pathways were induced by the dimethylphenoxyacetate, 3,5-Dimethylcatechol is not subject to metacleavage. The alternative ortho-eleavage is also unproductive and gives rise to (+)-4-carboxymethyl-2,4-dimethylbut-2-en-4-olide as a dead-end metabolite. High yields of this metabolite were obtained with the mutant Alcaligenes eutrophys JMP 134-1 which constitutively expresses the genes of 2,4-dichlorophenoxyacetic acid metabolism.Item Open Access Der Abbau von Modellstrukturen der Kohle: Stoffwechselweg des Dibenzofuran- und Fluorenabbaus(1991) Engesser, Karl-Heinrich; Strubel, Volker; Trenz, Stefan Peter; Rothe, Bernd; Schmid, Andreas; Knackmuss, Hans-JoachimSeveral microorganisms have been isolated degrading structural elements of coal like dibenzofuran. fluorene and biphenyl. Extensive investigation of the degradation pathways revealed a common mechanism of initial attack. Although catalyzed by different enzymes, all three substrates are converted to 3-phenyl-substituted catechols, which, after meta-cleavage are transformed to simple aromatic structures like salicylate, phthalate and benzoate. This ring cleaving enzymes have been cloned and are further analyzed after subcloning. Two different initial dioxygenases seem to be present in some strains cataIyzing ether cleavage of dibenzofuran and oxygenation of biphenyl respectively. Attempts are presently made to clone the first enzyme in order to produce higher yields of its optically active products. Some of these compounds have been characterized and may be of commercial value as fine chemicals.Item Open Access Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases(1992) Layh, Norman; Stolz, Andreas; Förster, Siegfried; Effenberger, Franz; Knackmuss, Hans-JoachimBacteria were enriched from soil samples, using benzylcyanide, α-methyl-, α-ethyl- or α-methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(–)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.Item Open Access Microbial degradation of biaryl structures: relationships between fluorene, dibenzofuran and biphenyl pathways(1992) Trenz, Stefan Peter; Strubel, Volker; Knackmuss, Hans-Joachim; Engesser, Karl-HeinrichInitial dioxygenation of fluorene by dibenzofuran degrading strains occurs in the unusual angular position. The resulting dihydrodiendiol is converted to 3-(2-carboxyphenyl)catechol by action of a dehydrogenase. This is a novel activity for a dehydrogenase causing a C-C-bond cleavage. After growth with dibenzofuran and biphenyl respectively two different initial dioxygenases are expressed. The first enzyme shows a broad substrate range, the second enzyme only converts biphenyl. Strains degrading fluorene, dibenzofuran and biphenyl may constitute a unique physiological group.Item Open Access Dioxygenolytic cleavage of aryl ether bonds: 1,2-Dihydro-1,2-dihydroxy-4-carboxybenzophenone as evidence for initial 1,2-dioxygenation in 3- and 4-carboxy biphenyl ether degradation(1990) Engesser, Karl-Heinrich; Fietz, Walter H.; Fischer, Peter; Schulte, P.; Knackmuss, Hans-JoachimA bacterial strain, Pseudomonas sp. POB 310, was enriched with 4-carboxy biphenyl ether as sole source of carbon and energy. Resting cells of POB 310 co-oxidize a substrate analogue, 4-carboxybenzophenone, yielding 1,2-dihydro-1,2-dihydroxy-4-carboxy-benzophenone. The ether bond of 3- and 4-carboxy biphenyl ether is cleaved analogously by initial 1,2-dioxygenation, yielding a hemiacetal which is hydrolysed to proto-catechuate and phenol. These intermediates are degraded via an ortho and meta pathway, respectively. Alternative 2,3- and 3,4-dioxygenation can be ruled out as triggering steps in carboxy biphenyl ether degradation.Item Open Access Metabolism of dibenzofuran and dibenzodioxin as model for 2, 3, 7, 8-tetrachlorodibenzodioxin degradation(1990) Strubel, Volker; Knackmuss, Hans-Joachim; Engesser, Karl-HeinrichWe demonstrated that fluorene and DBF are attacked hy strain DPO 1361 via an angular dioxygenation. A pathway for DBF degradation is presented, which inidicates an analogy to the pathway estahlished for diphenyl (1.4). Further investigations will have to show whether the same angular dioxygenation mechanism is involved also in the degradation of dibenzodioxin.Item Open Access Bacterial metabolism of side-chain-fluorinated aromatics: unproductive meta-cleavage of 3-trifluoromethylcatechol(1990) Engesser, Karl-Heinrich; Rubio, Miguel Angel; Knackmuss, Hans-JoachimSixteen bacterial strains capable of degrading alkylbenzenes and alkylphenols were directly isolated from soil and water. The degradation pathways are discussed. Alkylcatechols are almost exclusively cleaved via meta-ring fission. Meta-cleavage of 3-trifluoromethyl-(TFM)-catechol was observed with all strains at different rates although the reaction rates compared to catechol as a substrate varied considerably. All 2-hydroxy-6-oxohepta-2,4-dienoic acid hydrolases investigated showed strong binding of 7,7,7-trifluoro-2-hydroxy-6-oxohepta-2,4-dienoic acid, the ring fission product of 3-TFM-catechol. Turnover rates, however, were negligible indicating this compound to be a general dead-end metabolite during metabolism of TFM-substituted compounds via meta-cleavage pathways.Item Open Access Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one(1994) Trenz, Stefan Peter; Engesser, Karl-Heinrich; Fischer, Peter; Knackmuss, Hans-JoachimAngular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.Item Open Access Microbial metabolism of chlorosalicylates: accelerated evolution by natural genetic exchange(1986) Rubio, Miguel Angel; Engesser, Karl-Heinrich; Knackmuss, Hans-JoachimMethylsalicylate-grown cells of Pseudomonas sp. WR 401 cometabolized 3-, 4- and 5-substituted halosalicylates to the corresponding halocatechols. Further degradation was unproductive due to the presence of high levels of catechol 2,3-dioxygenase. This strain acquired the ability to utilize 3-chlorobenzoate following acquisition of genes from Pseudomonas sp. B 13 which are necessary for the assimilation of chlorocatechols. This derivative (WR 4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR 4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR 4016. The acquisition of this property was accompanied by concomitant loss of the methylsalicylate phenotype. During growth on 4- or 5-chlorosalicylate the typical enzymes of chlorocatechol assimilation were detected in cell free extracts, whereas catechol 2,3-dioxygenase activity was not induced. Repeated subcultivation of WR 4016 in the presence of 3-chlorosalicylate produced variants (WR 4016-1) which grew on all three isomers.