## 3 Results and Discussion
### Expression of the Mutants and Characterisation of Their Hydrolysis Profiles  
From the 35 predicted mutants, 24 mutants were successfully heterologously expressed and purified (Table 1 and Supplementary Figure S12). Afterwards, their hydrolytic activities were investigated by different pNP-esters. The evaluation of the different PCI_Lip mutants revealed major differences in both their overall activities and their chain length specificities (Figure 1). The mutants at position F91 in the putative lid domain showed overall lower activities towards pNPP. Expressed as the ratio between the activity towards pNPO and pNPP, the mutants showed a higher selectivity of 1.3 for F91T and F91N, and up to 2.1 for the F91L and F91H mutants, in comparison to the ratio of pNPO/pNPP for the WT of 0.9. Apart from that, the activity against all substrates increased for the F91L and F91T mutants, whereas it decreased for the F91H and F91N mutants. Given its higher overall activity and the discrimination of pNPP, the F91L mutant showed the most promising results. The \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Amino Acid** & **Mutant** & **Expression** & **Amino Acid** & **Mutant** & **Expression** \\ \hline \multirow{6}{*}{F91} & G & - & & I & x \\  & H & x & & P & x \\  & L & x & & R & x \\  & N & x & & Q & - \\  & T & x & & & F & - \\ \hline \multirow{6}{*}{F129} & A & x & \multirow{2}{*}{L302} & A & x \\  & C & x & & P & x \\ \cline{1-1}  & M & - & & & A & x \\  & Q & x & & H & x \\  & R & x & & M & x \\ \hline \multirow{6}{*}{S163} & H & - & & & N & x \\  & M & - & & R & x \\ \cline{1-1}  & P & - & & Y & x \\ \cline{1-1}  & V & - & & A & x \\ \cline{1-1}  & Y & - & & D & x \\ \hline \multirow{2}{*}{I245} & F & x & \multirow{2}{*}{1529} & E & - \\ \cline{1-1}  & W & - & & G & x \\ \cline{1-1}  & & & & W & x \\ \hline \hline \end{tabular}
\end{table} 
Table 1: PCI_Lip mutants with higher selectivity towards short- and medium-chain fatty acids predicted in silico. x = successful expression, - = expression was not possible as no protein band was visible in the SDS-PAGE after purification. mutation of F129, which is placed in the substrate channel, led to a drastic loss of hydrolytic activity, resulting in almost inactive enzymes. Unfortunately, no mutant at position S163 could be solubly expressed, indicating that this amino acid may be crucial for the correct protein folding. Interestingly, the I245F mutant did not lead to an improvement in the pNPO/pNPP ratio but showed a significant increase in hydrolytic activity towards pNPB, pNPV, and pNPH and an overall increased hydrolytic activity compared to the WT. Mutations at the L300, L302, and L305 positions led to mutants with an improved hydrolysis profile, indicating that this part of the substrate channel is crucial for the chain length specificity of PCI_Lip. The L300R mutant showed an improved pNPO/pNPP ratio of 1.8 and an overall increase in activity, while the L300P and L300G mutants resulted in inactive or non-specifically hydrolyzing enzymes. The L302G and L302P mutants both revealed a decrease in activity against all tested substrates of about 50% compared to the PCI_Lip WT. These mutants had a major impact on the specificity of PCI_Lip. They showed almost no activity against pNPO and pNPP while having their highest activity against pNPH for the L302G and against pNPV for the L302P mutant. L305R had only a minor impact on the specificity as its hydrolysis profile mostly resembled that of the WT. The L305H and L305Y mutants showed a significant increase in hydrolytic activity against pNPP, leading to unfavorable pNPO/pNPP ratios of 0.6 for L305H and of 0.2 for L305Y (WT pNPO/pNPP: 0.9). Simultaneously, the overall activity was increased for the L305H mutant, while that of L305Y was decreased. More expedient changes in the hydrolysis profiles were observed for L305A, L305M, and L305N. All three mutants revealed an increased pNPO/pNPP ratio of 1.9 for L305A, 1.7 for L305M, and 1.6 for L305N.