*3.3. Enzyme Activity Assays and Characterization of Recombinant ReCR*

The ReCR enzyme activity was measured by the reduction of NAD<sup>+</sup> or oxidation of NADH at 340 nm (ε<sup>340</sup> = 6.3 mM−<sup>1</sup> cm−1). Unless otherwise specified, the standard enzyme activity assay for the ketone reduction was performed at 60 ◦C in duplicate using the assay mixture (2.5 mL) containing 10 mM NBPO, 0.4 mM NADH, and 50 mM PIPES buffer (pH 6.0). The standard assay mixture (2.5 mL) for the alcohol oxidation at 50 ◦C contained 50 mM (*R*/*S*)-2-octanol, 0.4 mM NAD+, and 50 mM CAPSO buffer (pH 10.0). Unless stated otherwise, the reduction and oxidation reactions were initiated by the addition of 5 μg purified enzyme, respectively. One unit of activity was defined as the amount of enzyme that oxidized or reduced 1 μmol NADH or NAD<sup>+</sup> per minute under optimal pH and temperature. The protein concentrations of ReCR samples were determined using the Bradford reagent with bovine serum albumin as the standard protein.

The optimal temperature of ReCR activity was determined at a series of temperatures ranging from 25 to 70 ◦C using 50 mM PIPES buffer (pH 6.0) for NBPO reduction or 50 mM CAPSO buffer (pH 10.0) for (*R*/*S*)-2-octanol oxidation. The optimal pH of ReCR activity was determined over a range of pH from 5.5 to 11.0 at 60 ◦C for NBPO reduction or 50 ◦C for (*R*/*S*)-2-octanol oxidation. The buffers (50 mM) used were 2-(*N*-morpholino)ethanesulfonic acid (MES, pH 5.5–6.0), piperazine-1,4-bisethanesulfonic acid (PIPES, pH 6.1–7.5), Tris-HCl (pH 7.5–9.0), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO, pH 9.0–10.0), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS, pH 10.0–11.0). All the pH values of the buffers used were determined at 25 ◦C using a Mettler Toledo FE20 FiveEasy pH Meter (Mettler-Toledo (Schweiz) GmbH, Greifensee, Switzerland).

The thermostability of the ReCR was investigated by determining its residual activities when the enzyme samples were incubated at 35 ◦C, 55 ◦C, or 60 ◦C. To determine the stability in the presence of organic solvents, the enzyme was incubated with organic solvent at 35 ◦C for 3.5 h and then the residual activities were assayed for NBPO reduction. The determination of kinetic constants for ReCR was carried out using different substrates. The substrates were NBPO (0–20 mM), acetone (0–1 M), 2-propanol (0–70 mM), 2-octanone (0–30 mM), and (*R*/*S*)-2-octanol (0–20 mM). Apparent values of *K*m and *V*max were calculated using a non-linear regression curve fitting to the Michaelis-Menten equation with the software Origin 8.0 (OriginLab Corporation, Northampton, UK). Data of kinetic parameters present mean values ± SD from three independent experiments.

#### *3.4. Asymmetric Reduction of NBPO Catalyzed by Whole Cells of E. coli BL21(DE3)/pEASY-E2-recr*

The asymmetric reduction of NBPO was carried out using (*R*/*S*)-2-octanol or 2-propanol as a co-substrate for the coenzyme regeneration. In the case of 2-propanol as the co-substrate, the reaction mixture (5 mL) contained 0.5 M NBPO, 10% (*v*/*v*) 2-propanol, 0.4 mM NAD+, and 0.4 g wet cells in 50 mM Tris-HCl buffer (pH 8.0). In the aqueous/(*R*/*S*)-2-octanol biphasic system, the reaction mixture (5 mL) contained 1.5 M NBPO, 60% (*v*/*v*) (*R*/*S*)-2-octanol, 1.2 mM NAD+, and 1.2 g wet cells in 50 mM Tris-HCl buffer (pH 8.0). The reactions were carried out in a C76 Water Bath Shaker (New Brunswick, Edison, NJ, USA) at 35 ◦C and 300 rpm for 12 h.

The reaction mixture was extracted with 5 mL of ethyl acetate under strong vibration. The organic phase in the samples was separated by centrifugation and dehydrated with anhydrous sodium sulfate; then, 1 μL dehydrated sample was applied onto the injector (250 ◦C) for GC analysis. The reactants were determined with an Agilent 6890N (Santa Clara, CA, USA) gas chromatograph equipped with a chiral GC column (BGB174, 30 m × 250 μm × 0.25 μm). The temperature program for GC analysis was set as follows: 5 ◦C/min from 100 ◦C to 125 ◦C, hold 3 min; 2 ◦C/min to 140 ◦C, hold 8 min; 1 ◦C/min to 150 ◦C. The peak areas were quantitated using specific external standards. The standards NBPO, (*S*)-NBHP, and (*R*)-NBHP were purchased from Sigma-Aldrich Corporation (Shanghai, China). Retention times of the reactants were listed as follows: 26.997 min for NBPO, 28.452 min for (*S*)-NBHP, and 28.739 min for (*R*)-NBHP (Figure S1). Specifically, the (*S*)-NBHP peak was further determined by GC-MS analysis (Figure S2).

#### *3.5. Construction, Characterization, and Docking Analysis of ReCR Variant Y54F*

Site-specific mutagenesis was carried out by inverse PCR using native pEASY-E2-*recr* as a template and a pair of primers Y54F F1 (5 -TACACCTTCGGCCTTCCTCTCACGC-3 ) and Y54F R1 (5 -AAGGCCGAAGGTGTACTGCTCCTCG-3 ) under conditions as follows: denaturation, 95 ◦C for 2 min; 30 cycles of 95 ◦C for 20 s, 68 ◦C for 20 s, and 72 ◦C for 3 min; and the final extension, 72 ◦C for 8 min. The PCR product was digested at 37 ◦C for 2 h to digest the native template with the help of *Dpn* I. The digested product was directly transformed into *E. coli* BL21(DE3) competent cells. The positive recombinant cells were cultured at 37 ◦C and 200 rpm in LB medium with 100 μg/mL Amp. The recombinant cell named as *E. coli* Trans1-T1/pEASY-E2-*recr-mut* was selected by colony PCRs and the recombinant plasmid pEASY-E2-*recr-mut* was further extracted and verified by DNA sequencing (Sunny Biotechnology, Shanghai, China). Following the same procedure for the recombinant ReCR, the positive recombinant cell named as *E. coli* BL21(DE3)/pEASY-E2-*recr-mut* was obtained and the ReCR variant Y54F was purified for further characterization including kinetic parameters and catalytic performance in NBPO reduction.

The homology model of ReCR was built on the X-ray crystallographic structures of ADH-A from *Rhodococcus ruber* (PDB: 2XAA, resolution of 2.8 Å) by HHpred server [37]. Water molecules, ligands, and other hetero atoms (except the NAD<sup>+</sup> coenzyme and the zinc ion) were removed from the protein molecule. The coenzyme was remodeled as NADH. The charge of the catalytic zinc ion was assigned to +2, and the ligating side chain of Cys 38 was set as deprotonated and negatively charged. For the homology model of ReCR Y54F, the substitution of Tyr54 to Phe was introduced by FoldX [38]. A structure energy minimization of the proteins was performed to remove improper torsions of the side-chain conformation and correct the covalent geometry. The ligand molecule structures (NBPO and NADH) were directly drawn in ChemBioDraw and followed by an energy minimization. Global docking was performed using AutoDock Vina under the default docking parameters [39]. Point charges were initially assigned according to the AMBER03 force field [40], and then damped to mimic the less polar Gasteiger charges. Subsequently, local docking was executed to predict the binding energy and fine-tune the ligand placement in the binding site.
