**5. Conclusions**

In summary, significant increase of (*R*)-enantioselectivity in the (*E*/*Z*)-citral reduction was achieved by saturation mutagenesis of P76 and R330 in OYE2y. Remarkably, the variants P76M/R330H, P76G/R330H, and P76S/R330H exhibited full (*R*)-enantioselectivity in the reduction of (*E*)-citral or (*E*/*Z*)-citral. The variants with improved (*R*)-enantioselectivity usually came along with lower catalytic activities, indicating that the sites P76 and R330 were important for enantioselectivity as well as activity. In contrast to P76, R330 was relatively distant from active sites and its substitutions brought more beneficial impacts on enantioselectivity. Our results proved that it was reasonable to alter the enantioselectivity of OYE2y by saturation mutagenesis of key residue distant from active sites.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/6/1057/ s1, Table S1: The primer information of site saturation mutation of P76 in OYE2y; Table S2: The primer information of site saturation mutation of R330 in Oye2y; Figure S1: SDS-PAGE (12%) analysis of the purified OYE2y R330X variants; Figure S2: SDS-PAGE (12%) analysis of the purified OYE2y P76X variants; Figure S3: Gas chromatography analysis for standards (*S*)-citronellal (22.459 min), (*R*)-citronellal (23.067 min), (*Z*)-citral (29.164 min), and (*E*)-citral (30.398 min); Figure S4: Gas chromatography–mass spectrometry analysis for (*S*)-citronellal (A), (*R*)-citronellal (B), (*Z*)-citral (C), and (*E*)-citral (D) in the asymmetric reduction of (*E*/*Z*)-citral.

**Author Contributions:** Conceptualization, X.Y.; Data Curation, X.Y., M.Y. (Meilan Yu), and M.Y. (Meirong Ying); Formal Analysis, X.Y., M.Y. (Meilan Yu), and M.Y. (Meirong Ying); Funding Acquisition, X.Y.; Investigation, S.Y., M.H., R.W., S.M., F.C., and M.Z.; Supervision, Z.W.; Writing—Original Draft, X.Y.

**Funding:** This work was supported by the Natural Science Foundation of Zhejiang Province, China (No. LY17B020012).

**Conflicts of Interest:** The authors declare no conflict of interest.
