*2.1. Characterization of Recombinant ReCR*

The 1044-bp-long gene encoding ReCR was PCR-amplified from the genomic DNA of *R. erythropolis* WZ010 and over-expressed in *E. coli* BL21(DE3) in the form of the recombinant plasmid pEASY-E2-*recr*. The recombinant ReCR with C-terminal His-tag was subsequently purified by Ni-NTA chromatography. The gene *recr* encoded 348 amino acids with a deduced mass of 36.17 kDa, and the purified recombinant ReCR was verified with a single band of around 44 kDa by SDS-PAGE (Figure 1). The encoded amino acid sequence of ReCR displayed a 98% identity to that of PAR or alcohol dehydrogenase from *R. erythropolis* DSM 43297 (ReADH) [25–28], with five amino acids Arg67, Ser94, Lys110, Ser233, and Arg336 in ReCR different from Lys67, Asn94, Gln110, Lys233, and Gly336 in PAR or ReADH (Figure 2). The structure-related sequence alignment revealed that the enzyme belonged to the superfamily of zinc-containing alcohol dehydrogenases and had all conserved residues for the binding of catalytic and structural zinc ions [29]. It should be noted that the activity of the enzyme was severely inhibited by the exogenous zinc ion (Table S1), similar to what was observed with other zinc-containing alcohol dehydrogenases [24,30].

**Figure 1.** SDS-PAGE (12.5%) analysis of the purified recombinant ReCR. Lane 1, 2 μg purified ReCR with C-terminal His-tag; lane M, molecular weight marker. Coomassie Brilliant Blue R-250 was used to visualize the protein bands in the SDS-PAGE gel.

**Figure 2.** Structure-related sequence alignment between ReCR and its homologous proteins. 2XAA, PDB code of alcohol dehydrogenase from *Rhodococcus ruber* DSM 44541; PAR, alcohol dehydrogenase from *Rhodococcus* sp. ST-10 (GenBank accession No.: AB020760.3); ReADH, alcohol dehydrogenase from *R. erythropolis* DSM 43297 (GenBank accession No.: AY161280.1). The amino acid sequences of both PAR and ReADH are identical. Shown above the alignments are elements of the secondary structure of 2XAA. The numbering shown is from 2XAA. Red stars, putative catalytic residues; blue stars, residues for the coordination of structural zinc. Strictly conserved residues are highlighted with red boxes.

The recombinant ReCR was strictly NAD+-dependent, since the enzyme activity was not detectable when NADP(H) was used as a coenzyme. The effect of pH on the activity was investigated within the pH range of 5.5–10.5. The maximum activities for NBPO reduction and (*R*/*S*)-2-octanol oxidation were observed at pH 6.0 and 10.0, respectively (Figure 3A), indicating that ReCR-catalyzed oxidation/reduction was pH-dependent [24]. The optimal temperature was 60 ◦C for NBPO reduction and 50 ◦C for (*R*/*S*)-2-octanol oxidation (Figure 3B). The enzyme activity in NBPO reduction was stable at 35 ◦C, whereas the remaining activity decreased to 50% of the initial activity after heat treatment at 60 ◦C for 1.5 h or 55 ◦C for 6.5 h (Figure 4A), demonstrating that its thermostability was superior to the heat-sensitive enzyme KR-110 [4]. Among the tested organic solvents, 20% (*v*/*v*) 2-propanol drastically decreased the activity of ReCR, similar to the performance of PAR in the presence of >10% (*v*/*v*) 2-propanol [18]. In contrast to 20% (*v*/*v*) 2-propanol, the enzyme displayed higher stability after 3.5 h incubation with 40% (*v*/*v*) (*R*/*S*)-2-octanol (Figure 4B).

**Figure 3.** Effect of pH (**A**) and temperature (**B**) on the activity of recombinant ReCR. The relative activities of 100% represent 85.8 U/mg for NBPO reduction (solid symbols) and 88.3 U/mg for (*R*/*S*)-2-octanol oxidation (open symbols). The buffers 2-(*N*-morpholino)ethanesulfonic acid (MES, ), piperazine-1,4-bisethanesulfonic acid(PIPES, ), Tris-HCl (), and 3-(cyclohexylamino)-2 hydroxy-1-propanesulfonic acid (CAPSO, ◆) were used for the reduction reaction, while the buffers Tris-HCl (), CAPSO (), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS, ) were used for the oxidation reaction.

**Figure 4.** The stability of ReCR against heat (**A**) and organic solvents (**B**). Symbols: () for 60 ◦C, ( ) for 55 ◦C, () for 35 ◦C. The relative activity of 100% represents 85.8 U/mg for NBPO reduction. The enzyme was incubated with organic solvent (40% (*v*/*v*) (*R*/*S*)-2-octanol, 40% (*v*/*v*) 2-octanone, 20% (*v*/*v*) 2-propanol, or 20% (*v*/*v*) acetone) at 35 ◦C for 3.5 h prior to the stability test against organic solvent.

The substrate specificity of ReCR was tested using a set of alcohols and ketones (Table 1). Among the tested substrates, the enzyme exhibited the highest activities with 2,3-butanedione in the ketone reduction and (*R*/*S*)-2-octanol in the alcohol oxidation. The purified ReCR presented an activity of 85.8 U/mg towards NBPO reduction at pH 6.0 and 60 ◦C. Distinct from PAR and its variants [14], the activity of ReCR toward *N*-Boc-3-pyrrolidone reduction was relatively low. Particularly, the activity towards the oxidation of either (*S*)- or (*R*)-NBHP was not detectable at various temperatures (25–75 ◦C) and pHs (6.0–10.0), suggesting that the ReCR-catalyzed NBPO reduction was irreversible. A similar case was the secondary alcohol dehydrogenase SdcA from *R. erythropolis* DSM 44534 catalyzing the irreversible (*S*)-2-octanol oxidation [31]. The *K*<sup>m</sup> and *k*cat/*K*<sup>m</sup> values for NBPO were 1.74 mM and 35.98 s−<sup>1</sup> mM−1, respectively (Table 2). The *k*cat/*K*<sup>m</sup> value for (*R*/*S*)-2-octanol and 2-propanol was 13.04 s−<sup>1</sup> mM−<sup>1</sup> and 9.74 s−<sup>1</sup> mM−1, respectively, implying that the use of (*R*/*S*)-2-octanol or 2-propanol as a co-substrate could be feasible to regenerate NADH in the NBPO reduction.


**Table 1.** Substrate spectrum of recombinant ReCR against ketones and alcohols a.

<sup>a</sup> Data present mean values ± SD from two independent experiments. <sup>b</sup> Relative activity of 100% represents 85.8 U/mg for NBPO reduction at pH 6.0 and 60 ◦C; <sup>c</sup> Relative activity of 100% represents 88.3 U/mg for (*R*/*S*)-2-octanol oxidation at pH 10.0 and 50 ◦C.


**Table 2.** Kinetic parameters of recombinant ReCR a.

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