1. Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168
- Author
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Samir Bejar, Kameleddine Bouchaala, Wacim Bejar, Mounira Ben Farhat, Ameny Farhat-Khemakhem, Radhouane Kammoun, Ines Boukhris, Emmanuelle Maguin, Hichem Chouayekh, Université de Sfax, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Tunisian Government (Contrat Programme CBS-LMB, project of Valorization of Research Results 'Overproduction, formulation and assessment of the efficiency of a novel thermostable phytase as feed additive in poultry diets'), CMCU project 'Chouayekh/Maguin' (2007-2009) [07G0922], AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), This research was endorsed by the Tunisian Government (Contrat Programme CBS-LMB and the project of Valorization of Research Results 'Overproduction, formulation and assessment of the efficiency of a novel thermostable phytase as feed additive in poultry diets') as well as the CMCU project no 07G0922 'Chouayekh/Maguin' (2007-2009). The authors wish to express their sincere gratitude to Dr Thorsten Eggert, Mrs Rozenn Dervyn and Mr Amin Mrabet for their valuable collaboration. The authors would also like to thank Mr Maher Siala ELT supervisor and teacher of English at the Faculty of Sciences of Sfax for carefully proofreading the present paper., and Chouayekh, Hichem
- Subjects
0106 biological sciences ,Original ,[SDV]Life Sciences [q-bio] ,Biophysics ,Cloning vector ,Bacillus subtilis ,01 natural sciences ,Applied Microbiology and Biotechnology ,Microbiology ,law.invention ,03 medical and health sciences ,Plasmid ,law ,010608 biotechnology ,Bacillus subtilis 168 ,Phytase ,overexpression ,multimeric DNA forms ,experimental designs ,thermostability ,Yeast extract ,030304 developmental biology ,Thermostability ,0303 health sciences ,biology ,biology.organism_classification ,Biochemistry ,Recombinant DNA ,Heterologous expression - 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.
- Published
- 2012