Details

Autor(en) / Beteiligte

• Qian, Wen‐Zhuo
• Ou, Ling
• Li, Chun‐Xiu
• Pan, Jiang
• Xu, Jian‐He
• Chen, Qi
• Zheng, Gao‐Wei

Titel

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

Ist Teil von

• Chembiochem : a European journal of chemical biology, 2020-09, Vol.21 (18), p.2680-2688

Ort / Verlag

Germany: Wiley Subscription Services, Inc

Erscheinungsjahr

2020

Link zum Volltext

Quelle

Wiley Online Library - AutoHoldings Journals

Beschreibungen/Notizen

• 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.