A superior lipase for cheese production: A precisely medium-chain selective lipase from Pleurotus citrinopileatus expressed in Pichia pastoris.

The lipase derived from Pleurotus citrinopileatus (PCI-Lip) shows great potential for cheese production but could not achieve high-level expression in E. coli. Herein, we successfully expressed PCI-Lip extracellularly in Pichia pastoris X-33 (PpPCI-Lip), which exhibited a 472-fold increase in activity (kcat/Km = 5.99 × 105 M-1‧S-1) against p-nitrophenyl octanoate compared to E. coli. Scaled-up fermentation in a 5-L fermentor achieved a high volumetric activity of 5.94 × 105 U‧g-1, 3.17 times higher than shake fermentation. PpPCI-Lip demonstrated higher specificity for medium-chain fatty acids than commercial Palatase 20,000 L, releasing caprylic acid, capric acid, and lauric acid to enhance creamy flavors in cheese, as confirmed by GC-MS. Molecular dynamics simulations revealed that the lid region (Cys65-Ser98) governs substrate entry, enabling high hydrolysis selectivity for medium-chain triglycerides (C10:0) due to lower steric hindrance, while long-chain triglycerides (C18:0, C18:1) showed reduced efficiency due to steric resistance and aggregation effects.