U-DPhenol and 4-Dphenol were used to rule out carboxylation of phenol in the C1-position by a strictly anaerobic, defined mixed culture. By mass spectrometric analysis of deuterated phenol species and of benzoate, which were formed from U-Dphenol by D/H-exchange or by carboxylation from cell suspensions, it was shown that only one deuterium (D) from the aromatic nucleus was replaced with a least 97 efficiency. This excluded benzoate synthesis by carboxylation in the C1-position of phenol. Finally, carboxylation in thepara-position of phenol was demonstrated with 4-Dphenol by gas chromatography/mass spectroscopy of the products. Since direct measurement of phenol carboxylase activity was impossible due to a very active interfering decarboxylase activity, the optimal pH range and ion strength, as well as the requirement of cations in crude cell-free extracts was characterized by means of D/H-exchange from deuterated phenol.
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