TABLE 1.

Protocol for purification of benzoate-CoA ligase from T. aromaticaa

Purification stepVolume (ml)Protein (mg)Activity (μmol min−1)Sp act [μmol min−1 (mg of protein)−1]Yield (%)Purification (fold)
Cell extract (supernatant from centrifugation at 100,000 × g)12.7608
Ammonium sulfate precipitation (55%)9.345364.80.141001
Dialysis1342459.40.14911
DEAE-Sepharose34.568.330.00.44463.1
Source 30Q14.529.125.30.87396.2
Reactive green39.41.219.916.531118
  • a Benzoate was used as substrate for the assay. Purification started from 10.5 g (wet mass) of cells grown with 2-aminobenzoate and nitrate. Similar results were obtained when cells grown with benzoate and nitrate were used or when the enzyme was purified from cells grown aerobically on benzoate.