Construction of laccase green catalytic system based on amino acid hotspot mutation

In order to construct a green catalytic system for laccase,this study focused on laccase （CcLAC） from Coprinus cinereu,and a 3D model of CcLAC was established using homology modeling and AlphaFold2. Virtual saturation mutations were performed on amino acids within the T1 Cu 5 Å region of the laccase active site. The lignin model substrate guaiacol （GUA） was then molecularly docked with the mutants one by one and ordered according to affinity to obtain a mutant library. Guided by enhanced hydrophobicity of the active pocket surface, the mutant library was selectively simplified to identify hotspot amino acids and a specific mutation strategy. The CcLAC mutant plasmid was further constructed and transformed into Trichoderma reesei. Recombinant Trichoderma reesei transformants were successfully obtained through primary screening for hygromycin resistance and secondary screening using the color-changing zone method. The laccase activity produced by the mutant CcLAC_P415W reached 8.89 IU/mL, 1.88 times that of the wild type, and its thermostability was also significantly improved compared to the wild type.Additionally, molecular docking and high-performance liquid chromatography identified acetylsyringone and syringaldehyde as high-activity natural mediators.Further studies showed that the catalytic system composed of these two mediators and CcLAC_P415W significantly promoted the degradation of GUA.The constructed recombinant laccase-complex natural mediator system achieved an 85.63% GUA degradation rate.This study lays the foundation for further improvement of the laccase-mediator system and the development of novel green and environmentally friendly catalysts.