Engineering the Activity of Old Yellow Enzyme NemR-PS for Efficient Reduction of (E/Z)-Citral to (S)-Citronellol

The cascade catalysis of old yellow enzyme, alcohol dehydrogenase and glucose has become a promising approach for one pot, two-step reduction (E/Z)-citral to (S)-citronellol, serving as chiral with rose fragrance. During the multi-enzymatic catalysis, enzyme is responsible conjugated C=C introduction center, requiring high activity (S)-enantioselectiviy. Herein, improve from Providencia stuartii (NemR-PS) strict (S)-enantioselectivity, semi-rational design on its substrate binding pocket was performed through combination homology modeling, molecular docking analysis, alanine scanning iterative saturation mutagenesis. NemR-PS variant D275G/F351A improved obtained then purified characterization, obeying inhibition kinetics. Compared wild type, parameters Ki Kcat/Km were increased 39.79 mM 2.09 s−1mM−1 128.50 5.01 s−1mM−1, respectively. Moreover, maintained avoiding trade-off effect between enantioselectivity. Either or co-expressed Yokenella sp. WZY002 (YsADH) Bacillus megaterium (BmGDHM6). In contrast whole-cell biocatalyst co-expressing NemR-PS, that shortened reaction time 36 h 12 in 400 (E/Z)-citral. manner constant feeding, accumulated product concentration reached up 500 completely eliminate residual intermediate by-product, suggesting effectiveness protein engineering catalytic efficiency.