## 3 Results and Discussion
### Analysis of Mutants and Molecular Dynamic Simulations
Based on the docking analysis, L302G was confirmed as the best candidate at this position, where the overall profile of the binding pocket was not significantly altered as shown in Supplementary Figures S8 and S9, but the spatial arrangement of L305 was slightly altered, perhaps due to the reduced possibilities of hydrophobic interactions between the isobutyl groups of L302 and L305. Thus, the replacement of L by G directly reduces the relevant steric hindrances in the binding region. However, the activity against pNPP as the long-chain substrate was decreased, which might be an effect of less hydrophobic interactions in the L302G mutant compared to the WT enzyme. Similar effects are observed for the different isoforms of C. _rugosa_ lipases, where weaker interactions with the substrates in lipase 2 occur compared to lipase 1 or lipase 3. [PERSON] et al. [38] speculated that this phenomenon confers higher esterase behavior for lipase 2 compared to lipases 1 and 3, respectively. In contrast, the exchange of L305A caused an obvious change in the binding pocket of the homology model of PCI_Lip. Due to the exchange, which results in a smaller space requirement for the residue L305, no interactions between F129 and L300 could be formed (Supplementary Figures S6 and S7). Therefore, the space on the right part of the binding pocket, which was the main binding region in the WT enzyme, was reduced. In addition, F91 was removed from the binding pocket and turned towards the outer surface. These results were all beneficial in creating enough space in the left side of the binding pocket of the L305A mutant. The end of the lid, the residues L86-F95, was also affected by this mutation. L86-T89 were merged into a part of an \(\alpha\)-helix, which might tighten the lid structure, and twisted it out to the reaction medium, which at the same time enlarged the entrance region of this active pocket. However, after docking, no pNPP molecule was observed in the right position. As the hydrolysis of long-chain fatty acids from triglycerides caused off-flavors in the final cheese product, this mutant is highly interesting. To better understand the effects of the mutation, molecular dynamics simulations were performed with \"model\" homo triglycerides with fatty acids of chain lengths C6:0, C10:0, and C16:0, and the L305A mutant was compared to WT PCI_Lip. For this purpose, the changes of RMSD (Root Mean Squared Error) of the crucial distances between the carboxyl carbon atom (from different fatty acid chains in triglycerides) and the hydroxyl group oxygen of S213 in WT PCI_Lip or L305A mutant were analyzed (Supplementary Figures S18 and S19). Generally, the L305A mutant showed a higher binding affinity for the triglycerides than PCI_Lip WT, which was reflected by the number of suitable binding modes and residence time of fatty acid chains in the active center (Supplementary Tables S51 and S52). For the wild type, most fatty acids entering the active pocket were long-chain fatty acids (C16:0). After mutation, the number of medium-chain fatty acids (C6:0 and C10:0) entering the active pocket was increased markedly. The ratio of these three fatty acids was also improved, indicating a higher selectivity of L305A. This effect might be due to the reshaping of the active pocket generated by the L305A exchange. However, not only the selectivity was changed, based on the MD analysis, but also the binding time of the substrates was different (Supplementary Figures S20 and S21). Here, L305A was able to accommodate more fatty acid chains at the same time compared to PCI_Lip WT, possibly due to the longer time required for the interfacial activation of PCI_Lip WT, where the ligands were bound only in the later phase of the calculation.