**4. Conclusions**

The enzyme ReCR showed high specific activity, moderate thermostability, and strict (*S*)-stereoselectivity for asymmetric bioreduction of NBPO to (*S*)-NBHP. The NAD(H)-specific enzyme was active over broad pH and temperature ranges, and tolerated a higher concentration of organic solvents, offering greater flexibility in practical biocatalysis. Particularly, the reduction of NBPO to (*S*)-NBHP was irreversible, which was kinetically in favor of both coenzyme regeneration and formation of (*S*)-NBHP. The substitution of Tyr54 to Phe further improved the catalytic efficiency of ReCR including kinetic parameters and the productivity of (*S*)-NBHP. The *k*cat/*K*<sup>m</sup> values of ReCR Y54F for NBPO (49.17 s−<sup>1</sup> mM<sup>−</sup>1), (*R*/*S*)-2-octanol (56.56 s−<sup>1</sup> mM<sup>−</sup>1), and 2-propanol (20.69 s−<sup>1</sup> mM<sup>−</sup>1) were 1.37, 4.34, and 2.12 times higher than those of ReCR (35.98 s−<sup>1</sup> mM−1, 13.04 s−<sup>1</sup> mM−1, and 9.74 s−<sup>1</sup> mM<sup>−</sup>1), respectively. Furthermore, the (*S*)-NBHP yield was increased from 77.78% to 95.89% in the aqueous/(*R*/*S*)-2-octanol biphasic system when asymmetric reduction of 1.5 M NBPO was catalyzed for 12 h by whole cells of *E. coli* overexpressing ReCR Y54F instead of ReCR. Taken as a whole, ReCR variant Y54F has a great potential in the asymmetric synthesis of (*S*)-NBHP using (*R*/*S*)-2-octanol or 2-propanol as a co-substrate.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/23/12/3117/ s1, Figure S1: Gas chromatograph analysis for standards *N*-Boc-3-piperidone (A), (*S*)-*N*-Boc-3-hydroxypiperidine and (*R*)-*N*-Boc-3-hydroxypiperidine; Figure S2: Gas chromatograph-mass spectrometry analysis of (*S*)-*N*-Boc-3 hydroxypiperidine in asymmetric reduction of *N*-Boc-3-piperidone; Figure S3: The Michaelis-Menten kinetics of ReCR; Figure S4. The Michaelis-Menten kinetics of ReCR variant Y54F; Table S1: Effect of metal ions, EDTA, dithiothreitol and sodium iodoacetate on the activity of recombinant ReCR.

**Author Contributions:** Conceptualization, X.Y. and M.Y. (Meirong Ying); Data curation, X.Y. and M.Y. (Meilan Yu); Formal analysis, X.Y., F.C. and M.Y. (Meirong Ying); Funding acquisition, X.Y. and M.Y. (Meilan Yu); Investigation, J.Z., C.W., M.H., Y.J. and F.C.; Supervision, Z.W.; Writing – original draft, X.Y. and M.Y. (Meirong Ying).

**Funding:** This work was supported by the Natural Science Fundation of Zhejiang Province, China (No. LY17B020012) and the National Natural Science Fundation of China (No. 21405140).

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