## 3 Results and Discussion
### Analysis of Mutants and Molecular Dynamic Simulations  
At least three positions, F91, L302, and L305, modulate the chain length specificity. Therefore, these sites were further analyzed. F91 has a double function; on the one hand, as a crucial residue inside the putative lid domain; on the other hand, it covers the entrance of the active pocket with its phenyl ring side chain. In general, phenylalanine \begin{table}
\begin{tabular}{c c c c c c} \hline  & \begin{tabular}{c} **Protein** \\ **Concentration** \\ **[umol L\({}^{-1}\)]** \\ \end{tabular} & **Substrate** & \begin{tabular}{c} \(v_{\rm max}\) \\ **[umol min\({}^{-1}\) L\({}^{-1}\)]** \\ \end{tabular} & \begin{tabular}{c} \(K_{\rm M}\) \\ **[mmol L\({}^{-1}\)]** \\ \end{tabular} & \
\begin{tabular}{c} \(k_{\rm cat}\)/\(K_{M}\)** \\ **[s\({}^{-1}\)]** \\ \end{tabular} &
\begin{tabular}{c} \(k_{\rm cat}\)/\(K_{M}\)** \\ **[s\({}^{-1}\) mol\({}^{-1}\) L]** \\ \end{tabular} \\ \hline \multirow{4}{*}{WT} & \multirow{4}{*}{1.96} & pNPB & 50.12 & 0.647 & 0.43 & 658 \\  & pNPH & 78.89 & 0.549 & 0.67 & 1222 \\  & pNPO & 215.82 & 0.718 & 1.83 & 2555 \\  & pNPP & 103.52 & 0.022 & 0.88 & 40,177 \\ \hline \multirow{4}{*}{F91L} & \multirow{4}{*}{2.74} & pNPB & 115.89 & 0.786 & 0.71 & 898 \\  & pNPH & 119.80 & 0.504 & 0.73 & 1448 \\  & pNPO & 655.00 & 1.328 & 3.99 & 3005 \\  & pNPP & 136.65 & 0.042 & 0.83 & 20,024 \\ \hline \multirow{4}{*}{L302G} & \multirow{4}{*}{3.82} & pNPB & 77.53 & 0.756 & 0.34 & 447 \\  & pNPH & 223.05 & 1.214 & 0.97 & 801 \\  & pNPO & 42.82 & 0.706 & 0.19 & 264 \\  & pNPP & 13.87 & 0.013 & 0.06 & 4733 \\ \hline \multirow{4}{*}{L305A} & \multirow{4}{*}{4.60} & pNPB & 77.12 & 2.299 & 0.28 & 122 \\  & pNPH & 45.51 & 0.645 & 0.16 & 256 \\ \cline{1-1}  & pNPO & 97.42 & 0.735 & 0.35 & 481 \\ \cline{1-1}  & pNPP & 33.27 & 0.022 & 0.12 & 5598 \\ \hline \end{tabular}
\end{table} 
Table 2: Overview of the kinetic data of PCI_Lip WT and mutants F91L, L302G, and L305A against selected model substrates. Listed are the maximum velocity (\(v_{\rm max}\)), the Michaelis-Menten constant (\(K_{\rm M}\)), the turnover number (\(k_{\rm cat}\)), the catalytic efficiency (\(k_{\rm cat}\)/\(K_{M}\)), and the protein concentration in all kinetic experiments, which were used for the calculation of \(k_{\rm cat}\). in proteins has fewer rotamers compared to most other amino acids except for proline, tyrosine, and tryptophane and is therefore structurally more rigid compared to most other amino acids [36]. Moreover, in the PCI_Lip model, many loops were above F91, which further restricted the free space for structural changes of the F91 residue. In the calculated homology model in the equilibrium state, the phenyl ring is facing the substrate channel resulting in an enormous steric hindrance for binding substrates in the entry region of the channel. After mutation and virtual screening, F91L was confirmed as the best candidate (Supplementary Tables S1-S6 and Supplementary Figures S10 and S11), as further suitable binding options were identified for all chromogenic substrates, which is consistent with the determined activities towards pNP-esters of different chain lengths. For this variant, a shift of the highest activity from pNPP to pNPO was observed in the activity profile. The aromatic ring structure has been replaced by a short-chain branched amino acid that is still able to facilitate hydrophobic interactions with the carbon chains of the substrates while creating more space. This allowed the substrates to bind deeper in the binding pocket (Supplementary Figures S10 and S11). In Lip12 from _Yarrowia lipolytica_, which has no structural similarity with PCI_Lip, an exchange of F148L in the substrate channel could increase the catalytic efficiency for all tested substrates [37]. Within the substrate channel, the replacement of L302 and L305 had the greatest impact on the hydrolysis profiles.