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Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus : Effects of Thioredoxin Fusion Tag and Triton X-100

2011; Hindawi Publishing Corporation; Volume: 2011; Linguagem: Inglês

10.4061/2011/316939

2090-0406

Sylvia Maria Campbell Alquéres, Roberta Vieira Branco, Denise Maria Guimarães Freire, Tito Lívio Moitinho Alves, Orlando B. Martins, Rodrigo Volcan Almeida,

Electrochemical sensors and biosensors

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.