Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability

Catechol 2,3-dioxygenase from the thermophilic Bacillus thermoleovorans A2 was purified and characterized. The catechol 2,3-dioxygenase has a molecula r mass of 135000 Da and consists of four identical subunits of 34700 Da. On e iron per enzyme subunit was detected using atom absorption spectroscopy. Enzyme activity was not inhibited by EDTA, suggesting that the iron is tigh tly bound. Addition of hydrogen peroxide to the enzyme completely destroyed activity, indicating that the iron was in the divalent state. The isoelect ric point of the enzyme was 4.8. The enzyme displayed optimal activity at p H 7.2 and 70 degrees C. The half-life of the catechol 2,3-dioxygenase at th e optimum temperature was 1.5 min under aerobic conditions and 10 min in a nitrogen atmosphere. This stability of the enzyme is comparable to the stab ility of the enzyme from the mesophilic Pseudomonas putida mt-2. The stabil ity of the cloned enzyme in E. coli extracts was identical to the stability in wild-type extracts, suggesting that no stabilizing factors were present in Bacillus thermoleovorans A2 In whole cells the half-life of the enzyme at 70 degrees C was approximately 26 min, when protein synthesis was disrup ted by chloramphenicol; however, the activity remained constant when protei n synthesis was not inhibited. From these results we concluded that catecho l 2,3-dioxygenase from Bacillus thermoleovorans A2 is not particularly ther mostable, but that the organism retains the ability to degrade phenol at hi gh temperatures because of continuous production of this enzyme.