## 3 Results and Discussion
### Screening of Mutants with Improved Enzme Activity  
Initially, single mutant libraries for each residue in the S1 pocket were generated. From each SSM library, approximately 200 colonies were randomly selected to establish 95% coverage of the desired amino acids substitutions and screened [30; 36]. The qualitative colorimetric screening of mutant library was conducted against linalyl and \(\alpha\)-terpinyl acetates, since PbAcE showed lower activity towards two substrates compared to _tert_-butyl acetate. The reaction lasted until the wild type showed a color change from red to yellow, during which the changes of the 96-well plates were photographed every 5 min (Figure 2). We attempted to narrow down the positive mutants to only those that showed improved activity in both substrates. In all libraries, the top twelve positive colonies for each residue were selected and sequenced. The sequencing results for each residue were as follows, with the substituted amino acid and time of appearance in parentheses: Lys91 substituted with Ala (6), His (5), Ser (1); His93 with Pro (4), Asp (2), Ala (2), Gln (1), Val (1), Cys (1), Gly (1); and Tyr182 with Val (3), Thr (2), Glu (3), Met (2), Trp (2). Notably, most replacements were frequently occurring amino acids in AcEs of CE7 (Figure S2). The His and Ala at position 91 occurred more frequently than Lys, while His was the most commonly occurring amino acids at residue 93, followed by Pro, Ile, and Leu. Position 182 was occupied by smaller and more polar residues compared to that of Tyr. The single positive mutants exhibiting relatively higher activity compared to that of the wild-type were selected as follows: Ala, His, and Ser at Lys91; Pro and Asp at His93; and Glu, Val, Met, Thr, and Trp at Tyr182. To efficiently harness the potential synergistic effects \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Residues** & **Substrates 1** & **Location/Function** & **Mutability 2** \\ \hline Lys91 & GTB, GTH & S1 & 7 \\ Tyr92 & GTB, GTH & S1 & - \\ His93 & GTB, GTH & S1 & 5 \\ Gly94 & GTB, GTH & Oxyanion hole & - \\ Tyr95 & All & Oxyanion hole & - \\ Ser96 & LA, GTB, GTH & Oxyanion hole & - \\ Gly97 & GTH & Gate keeper & 6 \\ Asn98 & GTH & Gate keeper & 6 \\ Glu104 & GTH & Gate keeper & - \\ Tyr182 & GTH & S1 & 9 \\ Gly184 & All & Active site & - \\ Gln186 & All & Oxyanion hole & - \\ Phe210 & All & S0 & - \\ Pro225 & All & S0 & 5 \\ Thr276 & All & S0 & 5 \\ Cys277 & All & S0 & - \\ His303 & TA, LA, GTB & Catalytic triad & - \\ \hline \hline \end{tabular} 1 Abbreviations: TBA, _tert_-butyl acetate; TA, \(\alpha\)-terpinyl acetate; LA, linalyl acetate; GTB, glyceryl tributyrate; GTH, glyceryl trtihexanoate. 2 Data from HotSpot Wizard analysis. Values greater than six imply that the residues are located in a highly mutable position.  
\end{table} 
Table 1: List of residues involved in substrate binding based on covalent docking model. of the mutants, we sequentially combined mutations from each residue. The combination of individual positive mutants often results in an increase in enzyme activity [30; 38]. In this study, we introduced Pro and Asp mutations at His93 into three K91 mutants: K91A, K91H, and K91S. Colorimetric screening was performed as described above; however, no distinct changes were detected in the double mutants (Figure 3a). Of the double mutants, K91A/H93P and K91H/H93P showed slightly better activity compared to that of the other double mutants. Not surprisingly, Proline was the second most frequently occurring amino acid at position 93 (Figure S2). Subsequently, triple mutants were generated by introducing an additional mutation--Glu, Val, Met, Thr, and Trp at Tyr182--into the double mutant templates of K91A/H93P and K91H/H93P. The activity of the triple mutants was screened, revealing that the Y182E, Y182V, Y182M, Y182T, and Y182W variants in the K91A/H93P double mutant exhibited slightly higher or comparable activity to that of the wild type (Figure 3b).