15 Fakultätsübergreifend / Sonstige Einrichtung
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Item Open Access Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains(1986) Rubio, Miguel Angel; Engesser, Karl-Heinrich; Knackmuss, Hans-JoachimThe hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (5rarr10 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced over-production of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.Item Open Access Assemblage of ortho cleavage route for simultaneous degradation of chloro- and methylaromatics(1987) Rojo, Fernando; Pieper, Dietmar H.; Engesser, Karl-Heinrich; Knackmuss, Hans-Joachim; Timmis, Kenneth N.Genetic engineering is a powerful means of accelerating the evolution of new biological activities and has considerable potential for constructing microorganisms that can degrade environmental pollutants. Critical enzymes from five different catabolic pathways of three distinct soil bacteria have been combined in patchwork fashion into a functional ortho cleavage route for the degradation of methylphenols and methylbenzoates. The new bacterium thereby evolved was able to degrade and grow on mixtures of chloro- and methylaromatics that were toxic even for the bacteria that could degrade the individual components of the mixtures. Except for one enzymatic step, the pathway was fully regulated and its component enzymes were only synthesized in response to the presence of pathway substrates.Item Open Access Simultaneous degradation of chloro- and methylaromatics via ortho pathway by genetically engineered bacteria and natural soil isolates(1989) Engesser, Karl-Heinrich; Pieper, Dietmar H.; Rojo, Fernando; Timmis, Kenneth N.; Knackmuss, Hans-JoachimThe simultaneous bacterial metabolism of chloro- and methylaromatics via ortho- or metapathway, normally results in incomplete degradation and death of the organisms. This is caused by misrouting of central intermediates. i.e. substituted catechols into unproductive pathways and suicide inactivation of the key enzyme of meta pathway, (catechol 2,3-dioxygenase). The meta pathway proved to be definitely unsuited for productive metabolism of chloroaromatics. Therefore two strategies were used for simultaneous degradation of mixtures of chloro- and methylaromatics via ortho pathways: Methyllactons or certain mixtures of chloro- and methylaromatics were used as enrichment substrates, yielding strains which metabolized these compounds almost exclusively via the desired pathway. Alternatively relevant enzymes from five different catabolic pathways of three distinct soil bacteria were combined in a patchwork fashion generating a functional ortho cleavage route for methylaromatics coexisting with the ortho cleavage pathway of chloroaromatics.Item Open Access Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134(1988) Pieper, Dietmar H.; Reineke, Walter; Engesser, Karl-Heinrich; Knackmuss, Hans-JoachimOf eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Item Open Access Purification and characterization of 4-methylmuconolactone methylisomerase, a novel enzyme of the modified 3-oxoadipate pathway in the gram-negative bacterium Alcaligenes eutrophus JMP 134(1990) Pieper, Dietmar H.; Stadler-Fritzsche, Karin; Knackmuss, Hans-Joachim; Engesser, Karl-Heinrich; Bruce, Neil C.; Cain, Ronald B.4-Carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone) isomerase, transforming 4-methyl-2-enelactone to 3-methyl-2-enelactone, was purified from a derivative strain of Pseudomonas sp. B13, named B13 FR1, carrying the plasmid pFRC2OP. This plasmid contained the isomerase gene cloned from Alcaligenes eutrophus JMP 134, which uses 4-methyl-2-enelactone as a carbon source. The enzyme consists of a single peptide chain of Mr 40,000 as judged by SDS/PAGE. In addition to 4-methyl-2-enelactone, the putative reaction intermediate, 1-methyl-3,7-dioxo-2,6-dioxy-bicyclo[3.3.0]octane (1-methylbislactone), was a substrate for the enzyme, but kinetic data presented did not favour its role as a reaction intermediate. Isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides were neither substrates nor inhibitors. Possible reaction mechanisms are discussed.Item Open Access 3-Fluorobenzoate enriched bacterial strain FLB 300 degrades benzoate and all three isomeric monofluorobenzoates(1990) Engesser, Karl-Heinrich; Auling, Georg; Busse, Jürgen; Knackmuss, Hans-JoachimThe bacterial strain FLB300 was enriched with 3-fluorobenzoate as sole carbon source. Besides benzoate all isomeric monofluorobenzoates were utilized. Regioselective 1,2-dioxygenation rather than 1,6-dioxygenation yielded 4-fluorocatechol and minimized the production of toxic 3-fluorocatechol. Degradation of 4-fluorocatechol was mediated by reactions of ortho cleavage pathway activities. Chemotaxonomic and r-RNA data excluded strain FLB300 from a phylogenetically defined genus Pseudomonas and suggested its allocation to the alpha-2 subclass of Proteobacteria in a new genus of the Agrobacterium-Rhizobium branch.Item Open Access Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657(1988) Engesser, Karl-Heinrich; Cain, Ronald B.; Knackmuss, Hans-JoachimThe TOL plasmid-encoded enzymes of the methyl-benzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.