Optimizing the production of an α-(1→2) branching sucrase in [i]Escherichia coli[/i] using statistical design
| Autor: | Claire Moulis, Magali Remaud-Simeon, Marlène Vuillemin, Yannick Malbert, Sandrine Laguerre |
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| Přispěvatelé: | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), French National Research Agency [ANR-10-ALIA-0003], Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA) |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
0106 biological sciences
Time Factors [SDV]Life Sciences [q-bio] Glucansucrase Biology Biostatistics medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology law.invention Sucrase 03 medical and health sciences chemistry.chemical_compound Erlenmeyer flask Response surface methodology law 010608 biotechnology medicine Escherichia coli Inducer Lactose E. coli heterologous protein production 030304 developmental biology 0303 health sciences Expression vector Protein expression optimization Temperature General Medicine Culture Media chemistry Biochemistry biology.protein Box-Behnken design Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology Applied Microbiology and Biotechnology, Springer Verlag, 2014, 98 (11), pp.5173-5184. ⟨10.1007/s00253-014-5627-5⟩ Applied Microbiology and Biotechnology, 2014, 98 (11), pp.5173-5184. ⟨10.1007/s00253-014-5627-5⟩ |
| ISSN: | 0175-7598 1432-0614 |
| DOI: | 10.1007/s00253-014-5627-5⟩ |
| Popis: | Experimental design and Response Surface Methodology (RSM) were used to optimize the production of a dagger N-123-GBD-CD2, an alpha-(1 -> aEuro parts per thousand 2) branching sucrase previously reported as mainly produced in inclusion bodies. The a dagger N-123-GBD-CD2 encoding gene was cloned into two expression vectors in fusion with 6xHis tag or Strep tag II encoding sequences at 5' and 3' ends of the gene and expressed in five Escherichia coli strains. Three host-vector combinations were first selected on the basis of the amount of soluble enzyme produced. RSM with Box-Behnken design was used to optimize the expression conditions in an auto-inducible medium. Five factors were considered, i.e. culture duration, temperature and the concentrations of glycerol, lactose inducer and glucose repressor. The design consisted of three blocks of 45 assays performed in deep well microplates. The regression models were built and fitted well to the experimental data (R (2) coefficient > 94 %). The best response (production level of soluble enzyme) was obtained with E. coli BL21 Star DE3 cells transformed with the pET-55 vector. Using the predicted optimal conditions, 5,740 U L-1 (of culture) of soluble enzyme was produced in microtiter plates and more than 12,000 U L-1 (of culture) in Erlenmeyer flask, which represents a 165-fold increase compared to the production levels previously reported. |
| Databáze: | OpenAIRE |
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