A Deeper Insight into the Thermostability of a Novel Laccase Designed by Data-Driven Mining and Rational Engineering

Laccases, as multicopper oxidases, play a pivotal role in lignin degradation and hold broad industrial promise in biorefineries, bioremediation, textiles, and pulp and paper processing. However, their use is limited by poor stability under harsh operational conditions. Here, we designed a novel thermostable laccase (WCotA) from <i>Bacillus amyloliquefaciens</i> by combining data-driven mining with rational engineering. WCotA displayed remarkable thermostability and pH tolerance, with an optimal activity at 85 °C and a half-life of 1.0 h. To further improve thermostability, a combinatorial mutation library was constructed and screened, yielding mutant M2. M2 exhibited a prolonged half-life of 2.25 h at 85 °C and enhanced activity of 101.05 U/mg, outperforming the wild type. Structural and molecular dynamics analyses elucidated the mechanisms underlying its improved thermostability. This work provides a robust strategy for laccase discovery and engineering, advancing their sustainable application in industrial processes.