*2.2. Rational Design and Characterization of ReCR Variant Y54F*

For the in-depth characterization, attempts of rational design of ReCR were conducted to improve its activity. The ReCR homology model was built based on the X-ray crystal structure of ADH-A from *Rhodococcus ruber* (PDB: 2XAA). Sequence identity of ReCR towards ADH-A was 60%. The QMEAN and Z-score values were used for the quality evaluation of the models. The QMEAN and Z-score values of the ReCR homology model were 0.822 and 0.533, respectively, which indicated satisfactory quality. In Ramachandran Plot analysis, 91.5% of residues were located in a favorable region, and only 0.4% were found in the sterically disallowed region. This ReCR homology model was selected for subsequent docking studies.

Furthermore, substrate docking was employed to predict potentially beneficial amino acid positions on ReCR. Figure 5A shows that NBPO was ideally accommodated in the ligand binding pocket of ReCR composed by zinc ion, NADH, and Tyr54 (in the vicinity of the entrance to the active site). Similar to the binding mode of ADH-A with the substrate [29], the carbonyl oxygen atom of NBPO in ReCR was bound to the Zn2+ ion with a distance of 4.1 Å, and the carbonyl carbon atom was in close proximity to the C4-atom of NADH. Thus, the hydride was transferred onto the *re*-face of the carbonyl group, consistent with the strict (*S*)-enantioselectivity of ReCR. On the other hand, the bulky Boc group of NBPO was close to the hydroxyl group of Tyr54 (distance of 4.3 Å between the hydroxyl oxygen of Tyr and the tertiary carbon of the Boc group), which might cause a steric hindrance during the substrate binding (Figure 5). Therefore, Tyr54 was selected to be mutated to Phe.

**Figure 5.** Protein-ligand structures of ReCR with NBPO (**A**) and ReCR Y54F with NBPO (**B**). ReCR and ReCR Y54F are represented in cartoon format. Tyr54, Phe54, NADH, and NBPO are highlighted in sticks. The zinc ion is shown as a magenta sphere.

As anticipated, the substitution of Tyr54 to Phe significantly improved the catalytic performance of ReCR, implying that the amino acid residue at position 54 could be critical for the enzyme activity. In the ketone reduction, the *k*cat/*K*<sup>m</sup> values of ReCR Y54F for NBPO (49.17 s−<sup>1</sup> mM−1), acetone (1.47 s−<sup>1</sup> mM−1), and 2-octanone (53.21 s−<sup>1</sup> mM−1) were 1.37, 1.69, and 1.23 times higher than those of ReCR (35.98 s−<sup>1</sup> mM−1, 0.87 s−<sup>1</sup> mM−1, and 43.28 s−<sup>1</sup> mM−1), respectively (Tables 2 and 3). In the alcohol oxidation, the *k*cat/*K*<sup>m</sup> values of ReCR Y54F for (*R*/*S*)-2-octanol (56.56 s−<sup>1</sup> mM−1) and 2-propanol (20.69 s−<sup>1</sup> mM−1) were 4.34 and 2.12 times higher than those of ReCR (13.04 s−<sup>1</sup> mM−<sup>1</sup> and 9.74 s−<sup>1</sup> mM−1), respectively (Tables 2 and 3). Although the *K*<sup>m</sup> value of ReCR Y54F for NBPO (1.74 mM) was similar to that of ReCR, the *K*<sup>m</sup> values of ReCR Y54F for other tested substrates were lowered to a certain extent.


**Table 3.** Kinetic parameters of ReCR variant Y54F a.

<sup>a</sup> Data present mean values ± SD from three independent experiments. <sup>b</sup> ND, not detectable.

Consistently with kinetic parameters, the productivity of asymmetric bioreduction of NBPO was significantly enhanced when whole cells overexpressing ReCR Y54F instead of ReCR were used as biocatalyst (Table 4). In contrast to the free enzyme, the use of a whole-cell biocatalyst was chosen because of higher enzyme stability and simpler procedure of biocatalyst preparation [4,11,32]. Both 2-propanol and (*R*/*S*)-2-octanol were investigated as co-substrates for the NADH regeneration. In the presence of 10% (*v*/*v*) 2-propanol, the bioreduction of 0.5 M NBPO catalyzed by whole cells overexpressing ReCR Y54F gave a (*S*)-NBHP yield of 98.08% after 12 h, which was 1.34 times higher than that of ReCR (72.15%). The whole-cell biphasic system has been demonstrated to be effective at a higher substrate load, in which (*R*/*S*)-2-octanol instead of 2-propanol was used not only as co-substrate for coenzyme regeneration but also as the organic phase for the substrate reservoir and product sink [33,34]. In the aqueous/(*R*/*S*)-2-octanol biphasic system, the (*S*)-NBHP yield was increased from 77.78% to 95.92% when ReCR Y54F replaced ReCR in the whole-cell biocatalyst. The corresponding total turnover number value of 1199, the calculated space-time yield of 579.15 g L−<sup>1</sup> day−1, and the

remarkable stereoselectivity (*e.e.*<sup>p</sup> > 99.9%) together with the substrate concentration (up to 1.5 M) demonstrated a great potential of ReCR variant Y54F in the practical synthesis of (*S*)-NBHP.

**Table 4.** Asymmetric reduction of *N*-Boc-3-piperidone catalyzed by whole cells overexpressing ReCR or ReCR Y54F a.


<sup>a</sup> Data present mean values ± SD from two independent experiments. <sup>b</sup> Whole cells overexpressing ReCR or ReCR variant Y54F. <sup>c</sup> The *e.e.*<sup>p</sup> value (>99.9%) means that no (*R*)-NBHP peak was detected during GC analyses.
