Original Articles
Shagh Bandani, Valiollah Babae
Abstract
Lipoxygenase enzymes in eukaryotes play a significant role in the healing process. Based on recent studies, epidermal lipoxygenase enzyme extracted from amphibious is more effective in the healing process in comparison with human lipoxygenase. By the potential future significant effect in wound healing and tissue engineering, recombinant lipoxygenase axolotls (LOXe) production in E. coli was mentioned in this research. Clinical trial studies require sufficient amount of recombinant protein to generate. High level recombinant membrane protein LOXe over expresion in E. coli can be achieved by optimization of fermentation parameters such as induction conditions and culture medium. In this study, E. coli BL21 DE3 [pET21a-LOXe] (constructed by this research group) was used for enhanced r-LOX production. Production improvement was done in two stages: first evaluation of induction temperature, inducer concentration and medium culture effects on recombinant protein production in three levels by L9 array Taguchi experiment design approach, second optimization of carbon source amount and induction time in three levels by full factorial experiment deign in bioreactor, in turn. Experiments were done two times and responses (the amount of production of r-LOX) evaluated with design expert software. The obtained first step optimized conditions were induction temperature of 28 oC, inducer concentration 0.05 mg/l and TB medium culture. Optimized recombinant protein production and final dry cell weight under optimized conditions (glucose concentration of 10 g/l and induction time of OD600=5) were 18.8 and 3.54 g/l, respectively. The obtained values are of the highest amounts cell density and recombinant membrane proteins that has been reported up to now. The obtained high cell density cultivation in batch mode may be due to individual properties of LOXe and its effect on host cell growth rate.