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  • 1
    ISSN: 1432-072X
    Keywords: Thauera ; Toluene ; Benzyl alcohol ; Benzyl alcohol dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Toluene and related aromatic compounds are anaerobically degraded by the denitrifying bacterium Thauera sp. strain K172 via oxidation to benzoyl-CoA. The postulated initial step is methylhydroxylation of toluene to benzyl alcohol, which is either a free or enzyme-bound intermediate. Cells grown with toluene or benzyl alcohol contained benzyl alcohol dehydrogenase, which is possibly the second enzyme in the proposed pathway. The enzyme was purified from benzyl-alcohol-grown cells and characterized. It has many properties in common with benzyl alcohol dehydrogenase from Acinetobacter and Pseudomonas species. The enzyme was active as a homotetramer of 160kDa, with subunits of 40kDa. It was NAD+-specific, had an alkaline pH optimum, and was inhibited by thiol-blocking agents. No evidence for a bound cofactor was obtained. Various benzyl alcohol analogues served as substrates, whereas non-aromatic alcohols were not oxidized. The N-terminal amino acid sequence indicates that the enzyme belongs to the class of long-chain Zn2+-dependent alcohol dehydrogenases, although it appears not to contain a metal ion that can be removed by complexing agents.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-072X
    Keywords: Toluene ; Aromatic hydrocarbons ; Anaerobic metabolism ; Pseudomonas ; Benzoyl-CoA ; Benzyl alcohol ; Benzaldehyde ; Toluene methylhydroxylation ; Dehydrogenases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH3) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO2. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultanous adaptation: cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on tolucne; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.
    Type of Medium: Electronic Resource
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