title: Studying the expression of a lipase from Pyrococcus furiosus using response surfaces.
abstract: The need to find more stable catalysts has encouraged the study of naturally resilient enzymes, such as those found in extremophile organisms. In the present work, the influence of rare codons on the expression in Escherichia coli of the lipase Pf2001Δ60 from Pyrococcus furiosus was evaluated. Expression was carried out in two E. coli strains, BL21(DE3)pLysS and the rare tRNA supplemented Rosetta(DE3)pLysS. 3(2) factorial design was used to appraise the influence of temperature and inducer concentration on enzyme expression every hour for the 4-h expression period. Four response surfaces were constructed for each time, and the statistical parameters were evaluated. Lipase production was twice as high for Rosetta(DE3)pLysS than for BL21(DE3)pLysS. The factorial design indicated that optimal expression occurred at 30 °C after 4h, with lipase production of 240 U/L. The analysis of statistical parameters during the expression time showed that the velocity at which the enzyme was produced affected cell growth and maximum activity, with a higher speed leading to lower expression and cell growth. The presence of rare tRNAs prevented bottlenecks in lipase expression, and the experimental design was shown to be important for maximizing the production strategies and minimizing the metabolic load to which the host is subjected.
authors.affiliation: Laboratório de Microbiologia Molecular e Proteínas, Instituto de Química, Universidade Federal do Rio de Janeiro, Brazil.
authors.forename: Marcelo Victor Holanda
authors.initials: MV
authors.lastname: Moura
authors.forename: Leticia
authors.initials: L
authors.lastname: Dobler
authors.forename: Melissa Limoeiro Estrada
authors.initials: ML
authors.lastname: Gutarra
authors.forename: Rodrigo Volcan
authors.initials: RV
authors.lastname: Almeida
mesh_terms: D003001:Cloning, Molecular; D003062:Codon; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012343:RNA, Transfer / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013499:Surface Properties; D013696:Temperature
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D012343:RNA, Transfer; D008049:Lipase