Summary
The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K m of 0.17 mM and a V max of 1.05 U/mg and for ATP a K m of 0.16 mM and a V max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.
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Correspondence to: J. Winter
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Auburger, G., Winter, J. Purification and characterization of benzoyl-CoA ligase from a syntrophic, benzoate-degrading, anaerobic mixed culture. Appl Microbiol Biotechnol 37, 789–795 (1992). https://doi.org/10.1007/BF00174847
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DOI: https://doi.org/10.1007/BF00174847