Figure 3

B-carotene cleavage activity of mutants of aromatic residues located in binding cleft of BCMO1. A) Mutations of F51. B) Mutations of W454. C) Mutations of Y235. D) Mutations of Y326. Mid-log phase cultures of β-carotene producing E. coli transformed with pBAD/BCMO1 (+ control) or pBAD/mutant BCMO1 constructs were split in half; one half was induced with 0.02% L-arabinose, while the other was not. Both cultures were grown for 3 hours at 30°C in the dark and harvested. β-carotene was extracted and analyzed by RP-HPLC. To account for differences in the production of β-carotene in various cell cultures prior to induction we expressed β-carotene cleavage activity as (1-n) × 100%, where n is a ratio of β-carotene extracted from induced culture to β-carotene extracted from uninduced cell culture, normalized to protein concentration (+/-SE, N = 3).