title: The oxidation of mandelic acid and related compounds by bacterium NCIB 8250
Creators: name: Kennedy, Samuel I.T
Publisher: name: University of Glasgow
Year: 1967
Subjects: subject: Biochemistry

description: This thesis describes an investigation into the mechanism whereby the bacterium NCIB 8250 (which was formerly known as 'Vibrio 01') carries out the oxidation of mandelate and a number of related compounds. Bacterium NCIB 8250 was found to utilise L-mandelate, benzoylformate, benzyl alcohol, benzaldehyde or benzoate as sole source of carbon and energy for growth. 2-Hydroxy, 4-hydroxy, 3,4-dihydroxy and 4-hydroxy-3-methoxy derivatives of these compounds can also be utilised. The pathways of oxidation of all these substances were determined, in the first instance, by the technique of simultaneous adaptation, Washed cell suspensions, prepared from bacteria which had been grown on each aromatic compound in turn, were challenged in the Warburg apparatus with a large number of possible intermediates and analogues. The resulting patterns of oxygen utilisation indicated that the side chain of each L-mandelate or benzyl alcohol is oxidised to give the corresponding benzoate. L-Mandelate &gt; Benzoylformate &amp; Benzaldehyde Benzoate &gt; Benzyl Alcohol. The enzymes converting the L-mandelates or benzyl alcohols to the appropriate benzoates appear to be non-specific: indeed a variety of compounds which do not support growth, such as the 3-hydroxy substituted compounds are also metabolised to the corresponding benzoates but no further. The benzoic acids are then metabolised by a series of specific enzymes which are generally found only when cells have been grown in the presence of the corresponding substrate. Benzoate and 2-hydroxybenzoate are oxidised to catechol while 4-hydroxybenzoate and 4-hydroxy-3-methoxy-benzoate are converted to 3,4-dihydroxybenzoate. Catechol and 3,4-dihydroxy-benzoate then undergo ring fission. In order to substantiate the hypothesis that the enzymes converting the L-mandelates or benzyl alcohols to benzoates are non-specific these enzymes were examined in cell-free systems. Cell suspensions were subjected to ultrasonic disruption and, after centrifugation, the supernatant solutions were examined, Spectrophotometric assays were developed for L-mandelate dehydrogenase, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase. A suitably sensitive assay for benzoyl-formate decarboxylase was not obtained. It was found possible to characterise the enzymes by a number of parameters such as Km, Vmax and relative velocity with a range of substrates. Cell-free extracts were prepared after growth on a number of compounds and in all oases the kinetic properties of the enzymes were the same regardless of the growth substrate. Evidence obtained from experiments in which mixtures of substrates were added simultaneously ruled out the possibility of non-specific induction of several substrate-specific enzymes. The bacterium NCIB 8250 can therefore convert a large number of aromatic compounds to five key intermediates by means of just four enzymes. The thesis concludes with a discussion of the advantages to the bacterial cell of this type of specificity both in economy of cellular constituents and ecological status.