Journal
CHEMBIOCHEM
Volume 21, Issue 18, Pages 2680-2688Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202000196
Keywords
biocatalysis; chemical stability; cofactor regeneration; directed evolution; glucose dehydrogenase
Funding
- National Natural Science Foundation of China [21536004, 21672063, 21776085, 21871085, 31971380]
- Natural Science Foundation of Shanghai China [19ZR1472900]
- National Key Research and Development Program of China [2019Y-FA09005000, 2018YFC1706200]
- Fundamental Research Funds for the Central Universities [22221818014]
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Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDH(M0)) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH(M6) (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH(M6) was also more stable than BmGDH(M0) when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDH(M6) was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.
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