KnowVolution of a fungal laccase toward alkaline pH

  Copyright: © ChemBioChem KnowVolution of a fungal laccase toward alkaline pH: 3D model of variant Mal-M1 (L365E/L513M)

Congratulations to Catalina Novoa on her recent publication!

Given their ability to produce free radicals during the oxidation of phenolic and non-phenolic compounds, their catalytic versatility and eco-friendly mechanism, laccases are promising tools for a variety of biotechnological oxidative processes. Nowadays, commercial laccases are employed under mild pH conditions. Nevertheless, some potential applications are limited to pH. By increasing laccases alkaline tolerance, their potential applications could be broadened, such as in organic synthesis, bioremediation and pulp biobleaching. In this work, a fungal laccase from Melanocarpus albomyces (MaL) with increased alkaline tolerance (pH‑values ≥9.0) is reported. Through a Knowledge-gaining directed evolution (KnowVolution) campaign two beneficial substitutions were identified and combined in variant MaL-M1: L365 replaced by a negatively charged amino acid (E) and L513 by a hydrophobic amino acid (M), both located in close proximity of the T1Cu site. Characterization of the improved variant yielded a 3-fold increased kcat at pH 9.8 in comparison to the WT (kcat: MaL-M1: 6.0 ± 0.2 s-1; MaL-WT: 2.11 ± 0.07 s-1). Molecular dynamics simulations revealed that both substitutions synergistically work together to stabilize and improve laccase tolerance at higher pH.

This study was financed by the German Federal Ministry of Education and Research (BMBF) under the FuPol (Functionalization of Polymers) Alliance (FKZ: 031A227F) and by JARA‑HPC from RWTH Aachen University under the project RWTH0116.

Novoa, C., Dhoke, G. V., Mate, D. M., Martínez, R., Haarmann, T., Schreiter, M., Eidner, J., Schwerdtfeger, R., Lorenz, P., Davari, M. D., Jakob, F., and Schwaneberg, U. ChemBioChem, 2019. DOI:10.1002/cbic.201800807