DEGRADATION OF BENZOATE VIA BENZOYL-COENZYME-A AND GENTISATE BY BACILLUS-STEAROTHERMOPHILUS PK-1, AND PURIFICATION OF GENTISATE 1,2-DIOXYGENASE

The thermophilic Bacillus stearothermophilus PK1 utilized benzoate, 3- hydroxybenzoate, and gentisate as sole source of carbon and energy. 2- and 4-Hydroxybenzoate, 2,3- and 3,4-dihydroxybenzoate, and catechol d id not support growth. Degradation of benzoate proceeded via benzoyl-c oenzyme A (benzoyl-CoA) and gentisate. The inducible benzoyl-CoA ligas e converted benzoate but not 3-hydroxybenzoate to its coenzyme A thioe ster. Gentisate 1,2-dioxygenase from B. stearothermophilus PK1 was pur ified to homogeneity. The enzyme is presumed to be a homohexamer with a subunit molecular mass of 40 kDa. It showed maximal activity at 65-7 0 degrees C. After incubation for 80 min at 65 degrees C, 50% of the o riginal activity was lost. Gentisate 1,2-dioxygenase activity from str ain PK1 was strictly dependent on exogenously added Fe2+, and it was i nhibited by, metal-chelating agents, indicating an essential role of F e2+ in catalysis.