## 3 Results and Discussion
### Enzyme Activity and Structural Analysis
The triple mutant (K91A/H93P/Y182E) had a lower stability than the double mutant (K91A/H93P), owing to less hydrophobic interactions (Figures S5e and 5f). However, the mutations in this study did not seem to impact protein stability, since \(\Delta\Delta G\) was not significant [24]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Enzyme** & \(\mathbf{k_{cat}\) (\(\mathbf{s^{-1}}\))} & \(\mathbf{K_{M}\) (mM)} & \(\mathbf{k_{cat}\)/\(\mathbf{K_{M}}\) (mM\({}^{-1}\)s\({}^{-1}\))} \\ \hline Wild type & \(52.5\pm 0.9\) & 0.19 \(\pm\) 0.01 & 282.3 \\ \hline K91A & 53.5\(\pm\) 1.1 & 0.17 \(\pm\) 0.01 & 308.8 \\ \hline K91H & \(55\pm 0.8\) & 0.16 \(\pm\) 0.02 & 343.8 \\ \hline H93P & \(52.3\pm 0.9\) & 0.18 \(\pm\) 0.01 & 288.9 \\ \hline Y182E & \(55\pm 0.9\) & 0.17 \(\pm\) 0.01 & 323.1 \\ \hline Y182V & \(54\pm 0.9\) & 0.16 \(\pm\) 0.01 & 337.1 \\ \hline Y182M & \(52.5\pm 1.2\) & 0.16 \(\pm\) 0.01 & 328.1 \\ \hline Y182T & \(52.5\pm 1.0\) & 0.16 \(\pm\) 0.01 & 328.1 \\ \hline Y182W & \(55\pm 1.5\) & 0.16 \(\pm\) 0.01 & 343.8 \\ \hline K91A/H93P & \(50.5\pm 0.9\) & 0.17 \(\pm\) 0.02 & 297.1 \\ \hline K91H/H93P & \(51.4\pm 1.0\) & 0.17 \(\pm\) 0.01 & 302.4 \\ \hline K91A/H93P/Y182E & \(50.3\pm 0.7\) & 0.16 \(\pm\) 0.01 & 312.1 \\ \hline K91A/H93P/Y182V & \(52.5\pm 0.7\) & 0.17 \(\pm\) 0.01 & 308.1 \\ \hline K91A/H93P/Y182M & \(52.5\pm 0.9\) & 0.17 \(\pm\) 0.01 & 308.7 \\ \hline K91A/H93P/Y182T & \(54.3\pm 1.2\) & 0.17 \(\pm\) 0.01 & 319.3 \\ \hline K91A/H93P/Y182W & \(51.2\pm 1.1\) & 0.17 \(\pm\) 0.01 & 301.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The steady-state kinetic parameters of PbAcEs using \(p\)NP-C\({}_{2}\) as a substrate. Figure 5: Michaelis–Menten plot and covalent docking of \(p\)NP-C\({}_{2}\) onto PbAcE. (**a**) Michaelis–Menten plot of the steady-state kinetics of PbAcEs. The PbAcEs was assayed at 25 \({}^{\circ}\)C with increasing concentrations of \(p\)NP-C\({}_{2}\). (**b**) Two docked \(p\)NP-C\({}_{2}\) are displayed in triple mutant of PbAcE. The compound in yellow is a conformation when docked to wild-type PbAcE, while the one in pink is a conformation docked to triple mutants. Note that \(p\)NP-C\({}_{2}\) binds to the S0 pocket and shows no contact with the Lys91, His93, and Tyr182. We evaluated the residues of cold-active PbAcE that can prevent the proper binding of substrates using docking simulation and HotSpot Wizard software. PbAcE is an intriguing enzyme, since it presents broad substrate specificity and high activity at low temperatures. In particular, its characteristic of deacetylating CPC and related antibiotics render this enzyme a strong protein engineering candidate for enzyme-based pharmaceutical compound synthesis [1; 7; 14; 39]. Of its substrates, tertiary alcohol esters have low binding affinity to the substrate-binding pockets. PbAcE has a small acetyl binding pocket and large hydrophobic pocket, where the alcohol group of tertiary alcohol esters binds. To improve the suitability of the suRistrate-binding of the large pocket, we performed structure-guided engineering. Presently, there is growing number of publications supporting structure-guided protein engineering [7; 30; 35; 36; 37; 40; 41]. Based on docking simulation and hotspot analysis, we determined three suitable amino acids to be engineered. Structure-guided protein engineering combined with SSM or iterative saturation mutagenesis (ISM) has been widely used [30; 36; 37; 42; 43]. Usually in an ISM procedure, an improved mutant from the first-generation mutagenesis is chosen for the second-generation mutagenesis at other selected sites. After the first generation of SSM, we successfully harvested single mutants with improved activity. Instead of taking the second generation of SSM, we mainly combined the single best mutants using site-directed mutagenesis [36; 42; 43]. Contrary to other data showing the synergistic effect of introducing multiple mutations, our double and triple mutants did not exhibit higher activity than that of the corresponding single mutants.