Engineering a Highly Thermostable High-Redox Potential Laccase

Fungal high-redox potential laccases (HRPLs) are considered to be among the most relevant biocatalysts for industrial and environmental applications, although the lack of thermostable variants is hindering their practical implementation. In this letter, we describe the design of a highly thermostable HRPL. By introducing 27 stabilizing mutations identified in previous evolutionary campaigns focused on the HRPL from basidiomycete PM1, we have designed a variant with a half-life of thermal inactivation at 75 °C of 225 min, 32-fold superior to that of the parental enzyme. In addition, this variant displays remarkable stability at both acidic and basic pHs. The synergetic effect of the set of stabilizing mutations introduced is responsible for the improvements in thermal and pH stability, while maintaining the high-redox potential of this robust biocatalyst.