Figure 4
Effect of ultrafiltrate on activities of native (A) and recombinant wild-type CgPAD (B). Cell pastes of 6H2N-induced C. guilliermondii and recombinant E. coli were disrupted with glass beads in the extraction buffer. The fresh cell-free extracts obtained (5.8-7.5 mg protein ml-1) were ultrafiltered by centrifugation in an Amicon Ultra-15. Aliquots (10 μl to100 μl) of the ultrafiltrate were added immediately to the reaction mixture containing enzyme (0.95-ml final volume), incubated at 25°C for 20 min, and then the initial velocities toward substrates were measured by adding 50 μl of a 0.1 M substrate. The values were obtained from several separate experiments and are expressed as percentages, taking the activity toward FA (filled circle), PCA (open circle), and CA (filled triangle) of the untreated enzyme as 100%.