title: Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100.
abstract: In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX-PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX-PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100.
authors.affiliation: Laboratório de Biologia Molecular, Programa de Biotecnologia e Biologia Molecular, IBqM, UFRJ, 21941-902 Rio de Janeiro, RJ, Brazil.
authors.forename: Sylvia Maria Campbell
authors.initials: SM
authors.lastname: Alquéres
authors.forename: Roberta Vieira
authors.initials: RV
authors.lastname: Branco
authors.forename: Denise Maria Guimarães
authors.initials: DM
authors.lastname: Freire
authors.forename: Tito Lívio Moitinho
authors.initials: TL
authors.lastname: Alves
authors.forename: Orlando Bonifácio
authors.initials: OB
authors.lastname: Martins
authors.forename: Rodrigo Volcan
authors.initials: RV
authors.lastname: Almeida