## 3 Results and Discussion  
### In Silico Analysis of the Relevant Positions  
PCI_Lip belongs to the \(\alpha/\beta\)-hydrolase fold enzyme family. Like all known \(\alpha/\beta\)-hydrolases, it possesses the catalytically active core domain, a catalytic triad, which has been confirmed as S213, E339, and H449 by structural (Supplementary Figure S1) and sequence alignment (Supplementary Figure S2) [13; 31; 32]. The oxyanion hole signature is from GGGX-type, similar to that of _Candida rugosa_ lipase 2 (PDB code of the closed structure 1 GZ7) [32]. The substrate channel was predicted to be formed by the residues F91, F129, S163, L300, L302, A303, L305, I245, and I529, which were analyzed by investigating the cavities around the nucleophile S213 (Supplementary Figures S3 and S4). These residues are different compared to _C. rugosa_ lipase 2, which has a substrate-binding pocket formed by the residues L127, L132, and G450 (which corresponds to L131, M135, and A450 in PCI_Lip), and the entry of the hydrophobic channel is formed by the residues V296 and G344 (which correspond to I298 and H342 in PCI_Lip) [32]. In addition, the model of PCI_Lip has two regions (L86-F95 and L131-D138), which can be considered as covering lids on the entrance of the active pocket (marked in red in the Supplementary Figures S1-S4), while one lid-forming region is known for _C. rugosa_ lipase 2. In 1 GZ7, the lid domain is formed by a flap (residues P65-D94) that lies flat on the protein surface, and an important aromatic ring of F69 is buried in the hydrophobic pocket, while in PCI_Lip, this residue corresponds to L73, which is farther away from the predicted lid region in PCI_Lip. Interestingly, in PCI_Lip, F91 is potentially covering this role, as it is in the lid region, and the aromatic ring of this residue is located towards the substrate channel (Supplementary Figure S3). The lid domain mutagenesis/exchange can not only modulate the interfacial activity, it may also influence the activity, substrate specificity, and thermostability of an enzyme [14]. This was shown for _C. rugosa_ lipase 1 and 3 [33]. Since the substrate channel of PCI_Lip appears to be different from that of 1 GZ7, a focus was on identifying residues that interact with potential substrates in the hydrolysis of milk fat triglycerides, which contain more than 400 different fatty acids [3]. As the modeling of such complex fat is very cumbersome, the selectivity of PCI_Lip and mutants thereof was reflected by their affinities for model substrates with different lengths of carbon chains. For this purpose, pNP-esters of different chain lengths were docked inside the homology model of PCI_Lip (Supplementary Figures S4 and S5). Special attention was paid to the interaction of the substrates inside the binding pocket of PCI_Lip. The substrates pNPA, pNPB, pNPV, pNPH, and pNPO were able to fully enter the binding pocket of the WT enzyme, while the carbon chains of the substrates were bent, except pNPA, as the carbon chain is too short. A slight difference in the binding of the longer-chain chromogenic substrate pNPP was observed by in silico analysis, with the pNP-moiety located more at the solvent-accessible part of the substrate channel, possibly due to the higher steric hindrance of the carbon chain within the substrate channel. The binding analysis of the pNP-esters revealed that the residues inside the substrate channel could be divided into two regions: the left region containing the residues L300 and I529, and the right region consisting of F129, S163, I245, L302, A303, and L305. However, the A303 residue is the furthest away from the substrate channel cavity and orientated out of the channel and was therefore not mutated (Supplementary Figures S3 and S4), while S163 is also farther away in the original model based on the crystal structure of 1 GZ7 (Supplementary Figure S3), but after relaxing in the virtual water box, the substrate channel is a bit more open, and S163 is pointing into that channel. In _C.