Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents

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 nati...

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Published in:Chembiochem : a European journal of chemical biology 2020-09, Vol.21 (18), p.2680-2688
Main Authors: Qian, Wen‐Zhuo, Ou, Ling, Li, Chun‐Xiu, Pan, Jiang, Xu, Jian‐He, Chen, Qi, Zheng, Gao‐Wei
Format: Article
Language:English
Subjects:
Acetophenone
biocatalysis
Carbonyl compounds
Carbonyls
Catalysis
chemical stability
cofactor regeneration
Deactivation
Dehydrogenase
Dehydrogenases
Directed evolution
Evolution
Glucose
Glucose dehydrogenase
Hydrophobicity
NAD
Nicotinamide
Phenylbutyric acid
Reductases
Regeneration
Substrates
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Summary: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 (BmGDHM0) in the presence of large amounts of 1‐phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2‐fold increase in tolerance against 10 % (v/v) 1‐phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 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, BmGDHM6 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. Stability and tolerance: Our evolution generated highly chemical‐tolerant GDHs that would be useful for enzymatic reactions with high level of toxic chemicals. The developed chemically robust variants expand and enrich the GDH toolbox, facilitating its wider use for NAD(P)H regeneration in biocatalysis.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.202000196