High Output of a Trametes Laccase in Pichia pastoris and Characterization of Recombinant Enzymes

Abstract:

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21×10⁵u/L and 7.38×10⁴u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39×10⁵u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50℃ and at a pH range from 3 to 10. However, the specific activity of rLacDx (1,761u/mg) for ABTS was higher than that of rLacDe (1,122u/mg), and the apparent K_m value of rLacDx (427 μM) was less than that of rLacDe (604 μM).