title: Enhanced production of recombinant thermo-stable lipase in Escherichia coli at high induction temperature.
abstract: Thermostable microbial lipases are potential candidates for industrial applications such as specialty organic syntheses as well as hydrolysis of fats and oils. In this work, basic biochemical engineering tools were applied to enhance the production of BTL2 lipase cloned in Escherichia coli BL321 under control of the strong temperature-inducible λP(L) promoter. Initially, surface response analysis was used to assess the influence of growth and induction temperatures on enzyme production, in flask experiments. The results showed that temperatures of 30 and 45°C were the most suitable for growth and induction, respectively, and led to an enzyme specific activity of 706,000 U/gDCW. The most promising induction conditions previously identified were validated in fed-batch cultivation, carried out in a 2L bioreactor. Specific enzyme activity reached 770,000 U/gDCW, corresponding to 13,000 U/L of culture medium and a lipase protein concentration of 10.8 g/L. This superior performance on enzyme production was a consequence of the improved response of λP(L) promoter triggered by the high induction temperature applied (45°C). These results point out to the importance of taking into account protein structure and stability to adequately design the recombinant protein production strategy for thermally induced promoters.
authors.affiliation: Chemical Engineering Department, Federal University of São Carlos (UFSCar), Rodovia Washington Luiz, São Carlos, SP, Brazil.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Vélez
authors.forename: Antonio Carlos Luperni
authors.initials: AC
authors.lastname: Horta
authors.forename: Adilson José
authors.initials: AJ
authors.lastname: da Silva
authors.forename: Mônica Rosas da Costa
authors.initials: MR
authors.lastname: Iemma
authors.forename: Raquel de Lima Camargo
authors.initials: Rde L
authors.lastname: Giordano
authors.forename: Teresa Cristina
authors.initials: TC
authors.lastname: Zangirolami
mesh_terms: D001426:Bacterial Proteins; D019149:Bioreactors; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008049:Lipase; C428386:thermostable lipase
keywords: Fed-batch culture; Geobacillus thermocatenulatus; Lipase; Recombinant Escherichia coli; Thermal induction; λP(L) promoter