TABLE 2.
Copurification of the 3-oxoadipate enol-lactone-hydrolyzing and 4-carboxymuconolactone-decarboxylating activities of R. opacus 1CP
| Prepna | Vol (ml) | Protein (mg) | 3-Oxoadipate enol-lactonehydrolyzing enzyme
|
4-Carboxymuconolactonedecarboxylating enzyme
|
||||
|---|---|---|---|---|---|---|---|---|
| Total activity (U) | Sp act (U/mg) | Purification (fold) | Total activity (U) | Sp act (U/mg) | Purification (fold) | |||
| Cell extract | 142 | 256 | 157 | 0.61 | 1.0 | 145 | 0.57 | 1.0 |
| Q-Sepharose HP eluateb | 20 | 8.3 | 102 | 12.4 | 20.3 | 74 | 8.8 | 15.4 |
| Phenyl Superose eluatec | 4 | 0.15 | 9.1 | 61 | 100 | 8.0 | 53 | 93 |
a
The purification was performed basically as the first one but with different loading, with the buffer set to pH 7 and without dithiothreitol, and with changes in the chromatographic conditions as specified below.
b
The NaCl gradient for the Q-Sepharose High Performance (HP) step was 0 to 0.15 M over 10 ml and 0.15 to 0.5 M over 400 ml.
c
A phenyl Superose HR5/5 column was used with an (NH4)2SO4 gradient of 1 to 0.9 M over 3 ml and 0.9 to 0.5 M over 70 ml.