Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro

In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mg l⁻¹ laccase in yeast, together with a 1.4-fold increase in K_cat. Thus, the total activity is enhanced 5-fold for 2,2′-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6 mM copper, the highest activity value reached 30 U ml⁻¹ after a 3-day cultivation at a temperature of 30 °C_. In comparison with the wild type, the best mutant enzymatic properties (K_m for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 Å. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.