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AMB Express. 2012; 2: 10.
Published online Jan 26, 2012. doi:  10.1186/2191-0855-2-10
PMCID: PMC3305889
Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168
Ameny Farhat-Khemakhem,1 Mounira Ben Farhat,1 Ines Boukhris,1 Wacim Bejar,1 Kameleddine Bouchaala,1 Radhouane Kammoun,1 Emmanuelle Maguin,2 Samir Bejar,1 and Hichem Chouayekhcorresponding author1
1Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP "1177" 3018 Sfax, Tunisie
2INRA, UMR1319 Micalis, F-78350 Jouy en Josas, France; AgroParisTech, UMR Micalis, F-78350 Jouy en Josas, France
corresponding authorCorresponding author.
Ameny Farhat-Khemakhem: ameny2908/at/yahoo.fr; Mounira Ben Farhat: mounira.benfarhat/at/yahoo.fr; Ines Boukhris: i.boukhris/at/yahoo.fr; Wacim Bejar: wacim.bejar/at/yahoo.com; Kameleddine Bouchaala: boukameleddine/at/yahoo.fr; Radhouane Kammoun: radhouan.kammoun/at/cbs.rnrt.tn; Emmanuelle Maguin: emmanuelle.maguin/at/jouy.inra.fr; Samir Bejar: samir.bejar/at/cbs.rnrt.tn; Hichem Chouayekh: hichem.chouayekh/at/cbs.rnrt.tn
Received November 30, 2011; Accepted January 26, 2012.
Abstract
To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMβ1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMβ1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.
Keywords: Phytase, overexpression, Bacillus subtilis, multimeric DNA forms, experimental designs, thermostability
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