Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis.

Synthesis of ß-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating ß-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce ß-ionone, 3-hydroxy-4-oxo-ß-ionone, 3-hydroxy-ß-ionone, and 3-hydroxy-α-ionone in vitro. MnCCD1 could also cleave lycopene and ß-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and ß-ionone, respectively, in E. coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 °C. The addition of 10 % ethanol could increase enzyme activity at above 15 %. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0-1 mM). The Vmax for ß-apo-8'-carotenal was 72.5 U/mg, while the Km was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.

The effect of corticosteroids on pleurodesis induced by doxycycline in rabbits.

STUDY OBJECTIVE: To determine if the administration of systemic corticosteroids diminishes the effectiveness of the pleurodesis induced by the intrapleural injection of doxycycline. STUDY DESIGN: Thirty New Zealand white male rabbits were classified into three groups (n = 10 rabbits), all of which received doxycycline, 10 mg/kg intrapleurally, in a volume of 2 mL. Rabbits in the control group received only the intrapleural injection of doxycycline; the steroid-once group received triamcinolone, 0.8 mg/kg IM, 24 h before the intrapleural injection; and the steroid-weekly group received triamcinolone, 0.8 mg/kg IM, 24 h before the intrapleural injection and weekly over the next 3 weeks. All rabbits had a chest tube placed before the intrapleural administration of doxycycline and underwent pleural fluid aspiration twice daily. The rabbits were killed after 28 days, and the pleura and lungs were examined macroscopically and microscopically. RESULTS: The administration of corticosteroids resulted in both a significant decrease in the macroscopic adhesion score (p < 0.001) and a tendency toward a decreased microscopic fibrosis score (p = 0.056) after 28 days. Animals receiving weekly corticosteroid injections had lower scores than animals receiving only one injection. CONCLUSION: This study demonstrates that the administration of corticosteroids (triamcinolone, 0.8 mg/kg) 24 h before the intrapleural injection of doxycycline, 10 mg/kg, decreases the effectiveness of pleurodesis in rabbits. Weekly injections decreased the effectiveness even more. If these results can be extrapolated to humans, efforts should be made to minimize the administration of exogenous corticosteroids when pleurodesis is attempted.