Microbial formation of hydroxylated aromatic compounds from 4-chloro- and 4-nitrobenzoate

P. Groenewegen

    Research output: Thesisinternal PhD, WU


    <p>In the introduction of this thesis several aspects of the production of hydroxylated aromatic compounds are described. These compounds are applied in the production of pharmaceuticals, polymers, flavors and dyes, but their chemical synthesis is rather difficult in preparative organic chemistry. Therefore, biotechnological production might be an alternative approach, in particular in the case of specialty chemicals. It is discussed that an attractive way to accomplish the formation of a hydroxylated aromatic is by replacing a substituent on the aromatic nucleus by a hydroxyl group. In this research nitro and halogen substituted aromatic compounds were used as starting material for the biosynthesis of hydroxy-aromatics and the microbial degradation of these compounds is discussed extensively in <strong>chapter 1</strong> .<p>In the <strong>chapters 2</strong> and <strong>3</strong> the potential of the conversion of 4-chlorobenzoate to 4- hydroxybenzoate was investigated. In cell-free extracts of the coryneform bacterium NTB-1 a reaction was detected resulting in the formation of 4-hydroxybenzoate from 4-chlorobenzoate in the absence of oxygen. Surprisingly, although the hydroxyl donor in this reaction is water, with whole cells no conversion took place under anaerobic conditions. After this unexpected result the uptake system of 4-chlorobenzoate in this bacterium was studied in detail and a very specific energy-dependent uptake mechanism for 4-chlorobenzoate was detected. The uptake of 4- chlorobenzoate was shown to be coupled to the proton-motive force, suggesting a proton symport mechanism. The presence of this uptake system seemed to explain the lack of activity with whole cells under anaerobic conditions. After these findings, cells were permeabilized and anaerobic formation of 4-hydroxybenzoate was measured. Surprisingly, however, still no dehalogenase activity by whole cells was detected. But addition of alternative electron acceptors did result in a stoichiometrical conversion of 4-chlorobenzoate to 4-hydroxybenzoate. From these data it was concluded that apart from the energy-dependent uptake system also energy is required for regeneration of cofactors involved in the dehalogenase reaction. Examination of the mechanism involved in the dehalogenation of 4-chlorobenzoate revealed a novel mechanism in the degradation of halogenated aromatic compounds. In strain NTB-1 two enzymatic steps were involved in the conversion to 4-hydroxybenzoate: in the first step 4-chlorobenzoate was activated in the presence of ATP and CoA to 4-chlorobenzoyl:CoA. In the second step the 4- chlorobenzoyl:CoA ester was hydrolyzed to yield 4-hydroxybenzoate<p>In the second part of this thesis, enzymes involved in the conversion of nitro substituted to hydroxylated aromatic compounds were investigated ( <strong>chapters 4</strong> and <strong>5</strong> ).<p>After isolation of several organisms capable of degrading 4-nitrobenzoate, one strain designated NBA-10 was studied in detail. The degradation of 4-nitrobenzoate by <em>Comamonas acidovorans</em> NBA-10 followed a completely novel degradative pathway. In the first degradation steps, the 4- nitrobenzoate was reduced by a 4-NBA-reductase via 4-nitrosobenzoate to 4- hydroxylaminobenzoate. This reaction differed from published degradative pathways of nitro- aromatics involving release of ammonia. in such pathways formation of amino aromatic intermediates is often implied. However, in strain NBA-10 the involvement of 4-aminobenzoate as intermediate in the degradation of 4-nitrobenzoate was ruled out. The 4-NBA reductase was purified and it was demonstrated this hydroxylamine-forming enzyme was NADPH-dependent. The reductase displayed a narrow substrate specificity and it was not effected by flavine nucleotides. From these properties it was concluded that the reductase is quite distinct from other purified nitroaromatic reductases. In <strong>chapter 5</strong> it was shown the intermediate 4- hydroxylaminobenzoate is further metabolized to 3,4-dihydroxybenzoate. Formation of 3,4-dihydroxybenzoate was catalyzed by only one enzyme and also took place in the absence of oxygen. Purification of the enzyme revealed that apart from a reduced environment no cofactors were necessary for this reaction. The finding of this novel 4-hydroxylaminobenzoate degrading enzyme also elucidates various results found by others in the degradation of other nitroaromatic compounds.<p>In <strong>chapter 6</strong> it is discussed whether biotechnological processes are feasible in the formation of hydroxylated aromatics by replacing aromatic halogen or nitro groups. It was shown that anaerobical formation of 3,4-dihydroxybenzoate from 4-nitrobenzoate with strain NBA-10 can be achieved with resting cells provided a cosubstrate is present. Since this novel type of hydroxylaminobenzoate-degrading enzyme forms catechols in the absence of oxygen this type of bioformation might be interesting for the production of valuable catechols.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • de Bont, J.A.M., Promotor
    Award date15 Jan 1993
    Place of PublicationDelft
    Print ISBNs9789054850670
    Publication statusPublished - 1993


    • industrial microbiology
    • microbial degradation
    • derivatives
    • aromatic acids
    • carboxylic acids
    • benzoic acids

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