Optimization of Laccase PmL Secretion and Thermal Stability via Calculation-Guided Design for Mycotoxin Degradation.

Mycotoxin contamination causes an annual loss of approximately 30% of global grain production, threatening agricultural economies and food safety. Laccases offer a promising biodegradation route but suffer from low secretion yield and poor stability. This study characterized a novel Priestia megaterium laccase PmL and adopted a dual engineering strategy. A 222-sequence Pichia pastoris signal peptide library was constructed, and the optimal SP4 signal peptide elevated the extracellular activity of PmL to 2.94-fold of α-factor signal peptide, reaching 435.0 U/mL. Further PROSS computational design generated mutant SP4-M1, which simultaneously enhanced extracellular secretion (587.3 vs 147.9 U/mL) and thermostability (half-life at 80 °C: 423.7 vs 126.9 min) compared to PmL. Mycotoxin degradation assays confirmed SP4-M1 efficacy, achieving 99.5% degradation of aflatoxin B1 (AFB1) and 100% of zearalenone (ZEN). This systematic engineering strategy effectively optimizes the laccase performance, providing a reliable biocatalytic approach for mycotoxin contamination mitigation.