Characterization and Engineering of Basidiomycota-derived Lipases: Structural Insights and Optimization of hydrolytic Selectivity and Stability

In the production of piquant cheese varieties, such as Feta and Provolone, pregastric esterases (PGEs) are commonly used to generate characteristic piquant flavor profiles. These enzymes hydrolyze triglycerides present in milk fat during cheese ripening, releasing volatile free fatty acids that contribute to the distinctive aroma of these cheeses. These enzymes are traditionally derived from animal origin, and their use conflicts with the requirements for foods labeled as vegetarian, kosher, or halal. This interference raises the demand for enzymes with similar activity but of different origin. A lipase from the basidiomycete Pleurotus citrinopileatus (PCI_Lip) has been identified as a promising fungal substitute. To further adapt its catalytic properties to those of PGEs, a semi-rational protein engineering approach was applied to modify the chain length specificity for cheese production. Key residues influencing substrate specificity were identified in silico based on an AlphaFold3 model of PCI_Lip. In three rounds of mutagenesis, a small but smart library was generated. The library was screened for mutants with increased release of short to medium chain fatty acids and reduced release of long chain fatty acids. Alteration of substrate specificity was proven by changes in kinetic values compared to the wild type (WT) enzyme. Mutants with improved hydrolysis profiles were used for cheese production. The analysis of volatile free fatty acids and sensory evaluation showed that some mutants are suitable substitutes for PGEs. For a comprehensive characterization, stability, effects of immobilization, and regioselectivity of PCI_Lip were examined. The lipase was found to be highly thermostable with the potential to further increase its stability through suitable buffers and additives, as well as by immobilization. In the hydrolysis of triglycerides, the enzyme prefers the outer ester positions, therefore being characterized as an sn 1,3-selective lipase. Investigations on further Basidiomycota-derived lipases gave deeper insights into the structure and function of these enzymes. The characterization of a Pleurotus sapidus lipase (PSA_Lip) showed its thermostability and alkali tolerance. Besides activity on fatty acid esters, PSA_Lip was also active on ferulic acid esters. Sequence alignments with other hydrolases revealed that it does not belong to the big group of the α/β-hydrolases but displays a unique SGNH-like motif in the active site of the enzyme. The comparison of PCI_Lip with a lipase from Phlebia centrifuga (PCE_Lip) emphasized the role of the lid domain. A lack of this domain could be connected to reduced solubility and activity. The present work provides a comprehensive characterization of PCI_Lip and demonstrates strategies to optimize its catalytic properties and stability for various industrial applications. Through a combination of structural analysis, protein engineering, and immobilization techniques, the enzyme can be adapted to different parameters such as substrate selectivity and thermostability, making it applicable in various biotechnological applications. Comparative results with other Basidiomycota-derived lipases contribute to a better understanding of fungal hydrolases.

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Characterization and Engineering of Basidiomycota-derived Lipases: Structural Insights and Optimization of hydrolytic Selectivity and Stability

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