Computational and experimental engineering of a Pleurotus citrinopileatus lipases: Structural insights and functional optimization to adapt the hydrolytic profile for cheese applications

Mutants of a golden oyster mushroom lipase (<i>Pleurotus citrinopileatus</i>; PCI_Lip), were engineered to enhance hydrolysis profiles for cheese production. Key residues affecting activity were identified by SMME (Structure model based on AlphaFold3 prediction, followed by Molecular docking, Molecular dynamics, and Experimental validation) in three rounds of mutagenesis. Double mutants S163M + L302G and L302G + L305A showed significant improvements in photometric assays. The pNPH/pNPP ratios of 11.6 ± 1.1 and 10.4 ± 1.1 of both mutants, respectively, were improved compared to the wild type's 0.4 ± 0.1. Cheese made with S163M + L302G had a taste similar to the one prepared with a commercial enzyme in a descriptive testing, and both mutants demonstrated enhanced catalytic efficiency for short- to medium-chain fatty acids. These findings could be further confirmed by SPME-GC-MS analysis of the cheese samples. A structurally similar lipase from <i>Phlebia centrifuga</i> was investigated due to structural similarity with RMSD of 0.444 Å. The comparison of both lipases highlighted the key role of the lid domain in substrate specificity and affinity. This work advances the understanding of fungal lipases and their potential in food biotechnology.