## 3 Results and Discussion
### Enzyme Activity and Structural Analysis  
To further characterize the mutants, we expressed and purified eight single mutants (K91A, K91H, K91S, H93P, Y182E, Y182V, Y182M, and Y182W), two double mutants (K91A/H93P and K91H/H93P), and five triple mutants (K91A/H93P/Y182E, K91A/H93P/Y182M, K91A/H93P/Y182T, K91A/H93P/Y182V, and K91A/H93P/Y182W) (Figure S3). The enzymatic properties of these mutants were evaluated by measuring their relative activity against tertiary alcohols, such as linalyl acetate and \(\alpha\)-terpinyl acetate. The relative activities are presented in Figure 4a, with the corresponding colorimetric assay data displayed in Figure 4b. For linalyl acetate as the substrate, all mutants, except K91A/H93P/Y182W, exhibited higher activity than the wild type. Single mutants at the three different residues showed increased activities ranging between 10 and 30% relative to the wild-type PbAcE. The His93 mutations exhibited the smallest increase (10%) compared to mutations at Lys91 and Y182, which showed a 20-30% increase. Despite the introduction of multiple positive mutations, double and triple mutants only increased activity by approximately 10% relative to the wild type. For \(\alpha\)-terpinyl acetate, the relative activity enhancement pattern of mutant PbAcEs was similar to that observed with linalyl acetate, although the overall increment was generally lower. Single mutants demonstrated a greater increase in activity compared to that in double and triple mutants. Steady-state kinetics of PbAcE mutants were conducted using \(p\)NP-C\({}_{2}\) as a substrate to understand the impact of mutations on substrate affinity and catalysis. As shown in Figure 5a and Table 2, most of the mutants showed slightly lower \(K_{\text{M}}\) values for \(p\)NP-C\({}_{2}\) compared to that of the wild type, but the differences were not significant. This suggests that the mutations had little effect on kinetic parameters related to \(p\)NP-C\({}_{2}\) substrate. This is not surprising, as the mutations at three residues only enlarge the alcohol moiety binding pocket that \(p\)NP-C\({}_{2}\) does not occupy (Figure 5b). \(p\)NP-C\({}_{2}\) only binds the S0 pocket in the PbAcE. The \(p\)-nitrophenyl group has no interaction with the hydrophobic substrate binding pocket (S1), thus the mutation did not affect catalysis. Furthermore, we sought to understand the effects of the introduced mutations on the substrate-binding pocket size, substrate binding, protein stability, and conformation. To achieve this, we used the CavityPlus [26] and Dynamut [24] programs. The CavityPlus analysis demonstrated that the mutation improved the substrate-binding pocket (Figure 6). The pocket volume was 818.38 A\({}^{3}\) for the wild type, 858.38 A\({}^{3}\) for the K91A mutant, 884.88 A\({}^{3}\) for Y182E mutant, 855.38 A\({}^{3}\) for K91A/H93P mutant, and 974.62 A\({}^{3}\) for K91A/H93P/Y182E mutant. Notably, mutations in Lys91 and Tyr182 to smaller amino acids resulted in an enlarged binding pocket, potentially easing the accommodation of a large moiety of substrates. Figure 4: Enzymatic activity of PbAcEs. (**a**) Relative activities of mutant PbAcEs against linalyl acetate (black) and \(\alpha\)-terpinyl acetate (blue) compared to wild type. Relative activity of wild type was defined as 100%. (**b**) Representative image of the colorimetric assay at the specified reaction times. Mutations are indicated on the left side, and substrates are labeled across the top (Figure S4). The impact of mutation on protein stability and conformation was predicted using the DynaMut web server [24]. The protein stability and interatomic interaction were calculated and visualized (Figure S5). The mutation at Lys91 to Ala caused a reduced number of interactions because of the loss of positive charge and two methylene groups, which led to a slight destabilization (\(\Delta\Delta G\)\(-\)0.334 kcal/mol) but increased the flexibility (Figures S5a and 5b). The second mutation His93 to Pro slightly increased the stability and flexibility (Figures S5c and 5d).