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Engineering the Activity of Old Yellow Enzyme NemR-PS for Efficient Reduction of (E / Z)-Citral to (S)-Citronellol.
- Source :
-
Catalysts (2073-4344) . Jun2022, Vol. 12 Issue 6, pN.PAG-N.PAG. 14p. - Publication Year :
- 2022
-
Abstract
- The cascade catalysis of old yellow enzyme, alcohol dehydrogenase and glucose dehydrogenase has become a promising approach for one pot, two-step reduction of (E/Z)-citral to (S)-citronellol, serving as a chiral alcohol with rose fragrance. During the multi-enzymatic cascade catalysis, old yellow enzyme is responsible for the reduction of the conjugated C=C and the introduction of the chiral center, requiring high activity and (S)-enantioselectiviy. Herein, to improve the activity of the old yellow enzyme from Providencia stuartii (NemR-PS) with strict (S)-enantioselectivity, the semi-rational design on its substrate binding pocket was performed through a combination of homology modeling, molecular docking analysis, alanine scanning and iterative saturation mutagenesis. The NemR-PS variant D275G/F351A with improved activity was obtained and then purified for characterization, obeying the substrate inhibition kinetics. Compared with the wild type, the parameters Ki and Kcat/Km were increased from 39.79 mM and 2.09 s−1mM−1 to 128.50 mM and 5.01 s−1mM−1, respectively. Moreover, the variant D275G/F351A maintained strict (S)-enantioselectivity, avoiding the trade-off effect between activity and enantioselectivity. Either the enzyme NemR-PS or the variant D275G/F351A was co-expressed with alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) and glucose dehydrogenase from Bacillus megaterium (BmGDHM6). In contrast to the whole-cell biocatalyst co-expressing NemR-PS, that co-expressing the variant D275G/F351A shortened the reaction time from 36 h to 12 h in the reduction of 400 mM (E/Z)-citral. In the manner of substrate constant feeding, the accumulated product concentration reached up to 500 mM and completely eliminate the residual intermediate and by-product, suggesting the effectiveness of protein engineering and substrate engineering to improve catalytic efficiency. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 20734344
- Volume :
- 12
- Issue :
- 6
- Database :
- Academic Search Index
- Journal :
- Catalysts (2073-4344)
- Publication Type :
- Academic Journal
- Accession number :
- 157915246
- Full Text :
- https://doi.org/10.3390/catal12060631