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

Abstract EN

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.