title: Small-scale production of Burkholderia cepacia ATCC21808 lipase adapted to high-throughput screening.
abstract: 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.
authors.affiliation: UMR5504, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, CNRS, INRA, INSA, Toulouse, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Puech-Guenot
authors.forename: Vincent
authors.initials: V
authors.lastname: Lafaquière
authors.forename: David
authors.initials: D
authors.lastname: Guieysse
authors.forename: Laure
authors.initials: L
authors.lastname: Landric-Burtin
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Siméon
mesh_terms: 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
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D008049:Lipase