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

## TL;DR

Scientists improved a mushroom enzyme to better make cheese by changing its structure and testing its performance.

## Contribution

A novel approach combining computational modeling and experimental validation to optimize fungal lipase for cheese production.

## Key findings

- Double mutants S163M + L302G and L302G + L305A significantly improved hydrolytic activity compared to the wild type.
- Cheese made with the optimized enzyme had a taste similar to commercial enzyme-based cheese.
- The lid domain was identified as key to substrate specificity and affinity in fungal lipases.

## Abstract

Mutants of a golden oyster mushroom lipase (Pleurotus citrinopileatus; 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 Phlebia centrifuga 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.

•Basidiomycota lipase libraries with optimized hydrolysis profiles were investigated as alternatives to pregastric esterases•Comparative analysis of the lipases provided deeper insights into the role of the lid domain•Protein engineering was applied to tailor PCI_Lip to cheese-making requirements•Key residues influencing hydrolytic activity were identified and characterized

Basidiomycota lipase libraries with optimized hydrolysis profiles were investigated as alternatives to pregastric esterases

Comparative analysis of the lipases provided deeper insights into the role of the lid domain

Protein engineering was applied to tailor PCI_Lip to cheese-making requirements

Key residues influencing hydrolytic activity were identified and characterized