Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

• Published in: Biológia Vol. 66; no. 6; pp. 945 - 953
• Main Authors: Ayadi, Dorra Zouari, Kammoun, Radhouane, Jemli, Sonia, Bejar, Samir
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer-Verlag 01.12.2011; SP Versita; Versita
• Subjects: Bacillus stearothermophilus; Biomedical and Life Sciences; Cell Biology; culture media; cyclomaltodextrin glucanotransferase; E. coli; Enzymes; Escherichia coli; experimental design; gene overexpression; genes; Life Sciences; Microbiology; molecular cloning; optimization; Paenibacillus pabuli; Plant Sciences; recombinant CGTase; response surface methodology; screening; secretion; Section Cellular and Molecular Biology; signal peptide; temperature; Zoology; response surface methodology; secretion; recombinant CGTase; optimization
• ISSN: 0006-3088; 1336-9563
• DOI: 10.2478/s11756-011-0122-2

The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).