Small-scale production of Burkholderia cepacia ATCC21808 lipase adapted to high-throughput screening.
The screening of variant libraries of recombinant Burkholderia cepacia ATCC21808 lipase generated in Escherichia coli is limited by expression difficulties that are mainly due to the formation of inclusion bodies. To circumvent these difficulties and provide an efficient small-scale screening protocol, the gene encoding the lipase from B. cepacia was expressed in various expression vectors. With the pFLAG-ATS-Lip-Hp construct, expression of up to 6807 U/L of culture was possible in Erlenmeyer flasks. The production protocol was miniaturized in 96 deep-well plates, yielding 1300 U/L of lipase in fusion with the FLAG tag. With this protocol, the activity was determined in less than 10 min for a full plate, with a coefficient of variance of about 25%. For validation, 18 mutants constructed by site-directed mutagenesis on position Valine 266 were screened. Nice variations of activity were detected and found to be in agreement with those obtained in Erlenmeyer flask cultures. The protocol enabled the identification of 5 mutants showing enhanced activity toward para-nitrophenyl butyrate.
Puech-Guenot
Sophie
S
UMR5504, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, CNRS, INRA, INSA, Toulouse, France.
Lafaquière
Vincent
V
Guieysse
David
D
Landric-Burtin
Laure
L
Monsan
Pierre
P
Remaud-Siméon
Magali
M
D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D008049:Lipase