*3.2. Construction, Expression, and Purification of Recombinant Enzyme ReCR*

The gene *recr* was PCR-amplified from the genomic DNA of *R. erythropolis* WZ010 using forward and reverse primers: *recr*F1 (5 -ATGAAGGCAATCCAGTACAC-3 ) and *recr*R1 (5 -CTACAGACCAG GGACCACA-3 ). The PCR conditions were listed as follows: denaturalization, 94 ◦C for 4 min; 30 cycles of 94 ◦C for 30 s, 53.5 ◦C for 30 s, and 72 ◦C for 1 min; and the final extension, 72 ◦C for 10 min. According to TA cloning strategy from the instructions of the pEASY-E2 expression kit (TransGen Biotech Co., Ltd., Beijing, China), the PCR product was subcloned into the expression vector pEASY-E2 to form the recombinant vector pEASY-E2-*recr* with the C-terminal His-tag. The recombinant plasmid was then transformed into Trans1-T1 competent cells and the recombinant cells were cultured at 37 ◦C and 200 rpm in LB medium with 100 μg/mL ampicillin (Amp). The recombinant cell named as *E. coli* Trans1-T1/pEASY-E2-*recr* was selected by colony PCRs and the recombinant plasmid pEASY-E2-*recr* was further extracted and verified by DNA sequencing (Sunny Biotechnology, Shanghai, China).

The recombinant plasmid pEASY-E2-*recr* was extracted and then transformed into *E. coli* BL21(DE3) competent cells. The positive recombinant cell named as *E. coli* BL21(DE3)/pEASY-E2-*recr* was cultured at 37 ◦C and 200 rpm in LB medium with 100 μg/mL Amp. When the OD600 reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.3 mM, and the temperature was maintained at 20 ◦C. After 20 h incubation, the *E. coli* cells were harvested by centrifugation and the expression level was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following the same procedure in the study of 2,3-butanediol dehydrogenase from *R. erythropolis* WZ010 [24], the recombinant ReCR with C-terminal His-tag was purified to homogeneity by nickel affinity chromatography, desalted with 50 mM Tris-HCl (pH 8.0) by ultrafiltration, and stored at −20 ◦C for further characterization. The subunit molecular mass and purity of ReCR were verified by SDS-PAGE as described previously [36].
