Optimizing the production of an α-(1→2) branching sucrase in Escherichia coli using statistical design.
Experimental design and Response Surface Methodology (RSM) were used to optimize the production of ∆N123-GBD-CD2, an α-(1 → 2) branching sucrase previously reported as mainly produced in inclusion bodies. The ∆N123-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.
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France.
Marlène
M
Vuillemin
Yannick
Y
Malbert
Sandrine
S
Laguerre
Magali
M
Remaud-Siméon
Claire
C
Moulis
D056808:Biostatistics; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development*; D013393:Sucrase / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D003470:Culture Media; D013393:Sucrase