Microbial transformation of tetralin

J. Sikkema

    Research output: Thesisinternal PhD, WU


    <p>Biocatalytic oxidation of cyclic hydrocarbons has many potential applications in the production of fine chemicals. Especially regioselective hydroxylation of aromatics and the stereospecific formation of secondary alcohols is of interest for the pharmaceutical and flavoring industries. Hydroxylating enzymes are active under mild reaction conditions allowing the controlled transformation of less stable substrates and formation of easily oxidizable products (e.g., catechols). Furthermore, application of microorganisms for the removal of cyclic hydrocarbons from waste streams provides a highly versatile method for the removal of toxic cyclic hydrocarbons.<p>Major drawbacks in the application of biocatalysts, for these processes are the need for cofactor regeneration and the low stability as a result of inhibitory effects of the hydrocarbon substrates. The investigations described in this thesis have dealt with microbiological aspects of the design of a biocatalytic hydroxylation process. As a model for microbial oxy-functionalization, the dioxygenation of tetralin to 1,2,5,6,7,8-hexahydro- <em>cis</em> - naphthalene diol and the subsequent chemical rearrangement to 5,6,7,8-tetrahydro-1-naphthol has been studied. Tetralin provides a perfect model compound for specific hydroxylation since different sites of initial oxidative attack can be envisaged (Chapter 4). Moreover, different oxygenated derivatives of tetralin are of interest to pharmaceutical industries as precursors for the production of hormone-analogs, sedatives, and tranquilizers. Also for the production of fragrance compounds, oxygenated tetralins may be useful (Chapter 1 and 4). Special attention has been given to the mechanism of the toxicity of cyclic hydrocarbon substrates. In Chapter 2 literature data concerning inhibitory effects of cyclic hydrocarbons and other lipophilic compounds on microorganisms has been reviewed.<p><strong>Selection of suitable biocatalysts</strong> . Chapter 3 describes the procedure that has been followed to obtain microorganisms that are able to use tetralin as sole source of carbon and energy. Enrichment cultures on tetralin were set up with soil samples from polluted areas, and also cyclic hydrocarbon-utilizing strains from culture collections were tested. Initial attempts were unsuccessful, which was attributed to substrate inhibition. By lowering the concentration of tetralin in the incubation media, growth occurred in several enrichment cultures, but no pure strains were isolated. Eventually, a pure culture was isolated by supplying tetralin in subsaturating concentrations (lower than 125 μmol/liter). Initial studies on the inhibitory action of tetralin on this strain, <em>Arthrobacter</em> sp. strain T2, indicated that an aqueous concentration of approximately 100 μmol/liter). already impaired growth, whereas quantities above the saturation concentration (approximately 125 μmol/liter) fully inhibited growth of the starved cells. These findings were taken into consideration in new attempts to isolate tetralin-utilizing strains. Addition of tetralin via the vapor phase, thus limiting the aqueous concentration, resulted in the selection of another bacterium from an enrichment culture set up with soil from a land farming facility. Furthermore, four strains that were previously isolated on <em>o</em> -xylene, styrene, or mesitylene respectively were also shown to degrade tetralin. Alternatively, enrichment cultures were set up with tetralin added in a non-miscible, non-biodegradable, and non-toxic organic solvent (Fluorocompound 40) which limits the aqueous concentration by serving as a reservoir for the toxic substrate. From these enrichment cultures, two different strains were obtained that were able to use tetralin as sole source of carbon and energy.<p>In Chapter 4 a survey of initial oxidation steps that may be involved in the biotransformation of tetralin is presented together with experimental data on the accumulation of intermediates oxygenated intermediates from tetralin. The knowledge on the initial oxidative steps has been used to evaluate the potentialities of the selected strains. It appeared that five strains started with an initial oxidation of the benzylic carbon atom, resulting in the formation of a-tetralol and a-tetralone. Two strains exhibited aspecific oxidations yielding products characteristic of both oxidation of the aromatic and the alicyclic moiety. Only one strain, <em>Corynebacterium</em> sp. strain C125, degraded tetralin by initially oxidizing the benzene nucleus. The metabolic pathway of tetralin in <em>Corynebacterium sp.</em> strain C125 has been studied in detail. The results, which are presented in Chapter 5, show that the aromatic moiety is attacked by a dioxygenase at the carbon atoms proximal to the cycloalkane substituent. The cis-dihydro diol that is formed in this reaction is further metabolized via a dehydrogenase to 5,6,7,8-tetrahydro-1,2-dihydroxynaphthalene, which is a substrate for an extra-diol cleaving catechol dioxygenase. Acidrearrangement of 1,2,5,6,7,8-hexahydro- <em>cis</em> -1,2-naphthalene diol yielded the corresponding phenols, 5,6,7,8-tetrahydro-1-naphthol and 5,6,7,8-tetrahydro-2-naphthol, in a ratio of 1:6. The apparent preference for the 2-naphthol limits the feasability of this system for the formation of the desired fragrance compound, 5,6,7,8-tetrahydro-1-naphthol However, the specificities of the different tetralin- transforming strains enable the formation of some high-value precursors for the synthesis of pharamaceuticals (Chapter 1 and 4).<p><strong>Mechanism of the inhibitory action of tetralin and other cyclic hydrocarbons</strong> .<br/>From the results presented in Chapter 3 it is clear that tetralin has a deleterious effect not only on tetralin-utilizing strains but also on other organisms. From incubations with possible intermediates of tetralin metabolism it appeared that tetralin, and not an intermediairy reaction product, was responsible for the observed toxicity. Similar observations for other cyclic hydrocarbons suggested that the inhibitory action of these compounds resulted from interaction with the membrane(s) of microbial cells (Chapter 2).<p>In Chapter 6 the mechanism of the toxic action of tetralin on microorganisms has been investigated in intact cells of tetralin-utilizing and non-utilizing bacteria, as well as in liposomes. The results of these investigations indicated that tetralin was accumulated in the membrane, which lead to a significant increase in membrane surface area. Similar studies with other cyclic hydrocarbons, as described in Chapter 7, showed that in addition to the increase in surface area also an increased membrane fluidity was observed. The effective concentrations of the cyclic hydrocarbons necessary for disturbing membrane integrity decreased with increasing hydrophobicity (measured as partition coefficient in an octanol/water system). The hydrophobicity of the hydrocarbon compounds provides a good measure for the partition coefficients of these compounds between the aqueous environment and the membrane (Chapter 7). From the estimated membrane/buffer partition coefficients the actual concentrations of the different in the membrane could be calculated and related to their effects on the membrane properties.<p>As a result of the membrane expansion and increase in bilayer fluidity, the integrity of the membrane was impaired. Consequently, the passive permeability of the membrane to protons (ions) was increased and the activity of the membrane embedded proton-pump cytochrome c oxidase was reduced. As a result of an increased permeability for protons and impairment of proton-pumping activity, the proton motive force was dissipated and internal pH homeostasis was disturbed. The dissipation of the proton motive force may result in the depletion of metabolic energy, but lowering of the internal pH may lead to complete inactivation of enzymes.<p>In Chapter 8 some implications of the postulated mechanism of the toxic action of cyclic hydrocarbons have been discussed in relation to the application of microorganisms for the biotransformation of such compounds. Also some aspects of the adapation of the cells have been treated in connection with a general response of cells to stress. Finally, some methods to prevent deleterious effects of cyclic hydrocarbons have been discussed in view of the proposed toxicity mechanism.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • de Bont, J.A.M., Promotor
    • Poolman, B., Promotor, External person
    Award date24 May 1993
    Place of PublicationS.l.
    Print ISBNs9789054851257
    Publication statusPublished - 1993


    • derivatives
    • aromatic compounds
    • naphthalene
    • industrial microbiology
    • microorganisms
    • biochemistry
    • metabolism
    • synthesis
    • oxygenases

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