Qian Wen-Zhuo, Ou Ling, Li Chun-Xiu, Pan Jiang, Xu Jian-He, Chen Qi, Zheng Gao-Wei
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China.
Chembiochem. 2020 Sep 14;21(18):2680-2688. doi: 10.1002/cbic.202000196. Epub 2020 Jun 4.
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 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH was also more stable than BmGDH 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 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.
葡萄糖脱氢酶（GDH）是合成生物化学中驱动烟酰胺（NAD(P)H）再生的常用工具。越来越多的合成生物反应在含有大量有机助溶剂或疏水底物/产物的介质中进行，这常常会使天然酶变性，包括那些用于辅因子再生的酶。在这项工作中，我们试图通过定向进化提高巨大芽孢杆菌GDH（BmGDH）在大量苯乙醇存在下的化学稳定性。在所得突变体中，BmGDH（Q252L/E170K/S100P/K166R/V72I/K137R）对10%（v/v）苯乙醇的耐受性提高了9.2倍。此外，当暴露于苯乙酮、2-氧代-4-苯基丁酸乙酯和（R）-2-羟基-4-苯基丁酸乙酯等疏水且能使酶失活的化合物时，BmGDH也比野生型BmGDH更稳定。与光滑念珠菌羰基还原酶偶联后，BmGDH成功用于失活的2-氧代-4-苯基丁酸乙酯的不对称还原，烟酰胺辅因子的总周转数为1800，因此使其具有商业应用吸引力。总体而言，化学稳定性强的GDH的进化促进了其作为生物催化中NAD(P)H再生通用工具的更广泛应用。
