Isolation of a new anaerobic bacterium transforming phenol to benzoate and purification of the 4-hydroxybenzoate decarboxylase

Aromatic compounds are the second largest group of natural products. Some of them are mutagenic, teratogenic and/or carcinogenic. Especially, phenol and related compounds have a commercial importance since they are part of the aromatic compounds that are most widely used in modern world. Microbial degradation of aromatic compounds is the best solution of those contamination problem and more acceptable way up to now.; Anaerobic microbial degradation of aromatic compounds has received much attention over the past decade. This is mainly due to the fact that anaerobic biodegradation is more advantageous than aerobic biodegradation on an economic point of view. Once the aromatic substrate is anaerobically transformed to a simple aromatic intermediate, such as phenol or benzoic acid, the reduction occurs and converts the aromatic ring to an alicyclic ring which is then hydrolytically cleaved and gradually mineralized.; A new strain carboxylating phenol from a methanogenic consortium has been isolated under anaerobic conditions during this study. It is the first pure culture isolated from a methanogenic consortium for its ability to carboxylate phenol to 4-hydroxybenzoate and dehydroxylate 4-hydroxybenzoate to benzoate. This was achieved by treating a heat-treated methanogenic consortium with antibiotics followed by dilution to eliminate the four Clostridium strains which was without effect on the phenol transformation activity. Isolated strain 6 transformed phenol or 4-hydroxybenzoate in presence of proteose peptone. The new strain 6 was characterized. It is a flagellated rod, 0.6 $m$m width and a Gram-variable bacterium. It has a doubling time of 10 to 11 hours in the presence of phenol. The result of 16S rRNA sequence analysis showed that strain 6 was most similar to that of Clostridium species, with homology ranging from 80 to 86%.; The purification and characterization of a reversible 4-hydroxybenzoate decarboxylase from a coculture constituted of strain 6 and 7 is also described in this thesis. The reversible activity of the 4-hydroxybenzoate decarboxylase was responsible for the carboxylation of phenol to 4-hydroxybenzoate. The molecular mass of the enzyme was estimated to 420 kDa and consisted of four identical subunits of 119 kDa. The optimum temperature for decarboxylation had a wide range between 15–45°C and the optimum pH was 5.0–6.5. The pI of the enzyme was 5.6. The enzyme also catalyzed the decarboxylation of 3,4-dihydroxybenzoate. The K_m value obtained for 4-hydroxybenzoate was 4.3 mM and V_m was 139 $m$mol/min/mg at pH 6.5 and 37°C. No stimulation of the activity was observed upon addition of ATP. Phenol phosphorylation was not implicated in phenol or 4-hydroxybenzoate transformation. The N-terminal amino acid sequence of the purified enzyme was shown to be very similar to a pyruvate-flavodoxin oxidoreductase (nif J gene product).; These results give a better understanding of the microorganisms and of the enzyme implicated in a first step of phenol transformation. This knowledge is valuable for the eventual optimization of the anaerobic treatment of phenolic compounds.