90315-82-5Relevant articles and documents
Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents
Qian, Wen-Zhuo,Ou, Ling,Li, Chun-Xiu,Pan, Jiang,Xu, Jian-He,Chen, Qi,Zheng, Gao-Wei
, p. 2680 - 2688 (2020)
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.
Purification and characterization of carbonyl reductase from Candida krusei SW 2026 involved in enantioselective reduction of ethyl 2-oxo-4-phenylbutyrate
Li, Ning,Ni, Ye,Sun, Zhihao
, p. 190 - 197 (2010)
Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is widely used as a key chiral building block in the synthesis of a class of angiotensin-converting enzyme (ACE) inhibitors. A highly enantioselective carbonyl reductase responsible for the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) was identified and characterized from Candida krusei SW 2026. The enzyme was purified to homogeneity through three chromatography columns. The relative molecular mass of the enzyme was estimated to be around 45,500 by gel filtration and 46,000 by SDS-polyacrylamide gel electrophoresis. The enzyme yielded (R)-enantiomer product and utilized NADPH as the cofactor. The purified enzyme exhibited maximum activity at pH 6.0 and 30 °C, and retained over 80% of its activity over an acidic pH range of 4.5-7.0. The maximum reaction rate (V max) and apparent Michaelis-Menten constant (Km) for OPBE and NADPH were 18.7 μmol/(min mg) protein and 0.319 mmol, 14.9 μmol/(min mg) protein and 0.306 mmol, respectively.
Structure-guided evolution of carbonyl reductase for efficient biosynthesis of ethyl (: R)-2-hydroxy-4-phenylbutyrate
Chen, Chao,Chen, Lulu,Deng, Jian,Kuang, Yuyao,Lin, Jinping,Tang, Wen,Wang, Hualei,Wei, Dongzhi,Yin, Bo
, p. 7512 - 7522 (2020/11/27)
Ethyl (R)-2-hydroxy-4-phenylbutanoate ((R)-HPBE) is an important versatile intermediate for the synthesis of angiotensin-converting enzyme inhibitors. Herein, a structure-guided rational design was adopted to improve the catalytic performance of carbonyl reductase from Gluconobacter oxydans (GoCR) for efficient production of (R)-HPBE at high substrate loading. To enhance the catalytic performance of GoCR, three sites (Cys93, Ile187 and Trp193) were identified based on a computational approach. Through single-site and cooperative mutation at these three sites, four variants with simultaneous increase in stereoselectivity and catalytic efficiency were obtained. Variants mut-W193L, mut-W193L/C93I, mut-W193L/I187L and mut-W193L/C93I/I187L exhibited 9.8-to 37.0-fold increase in catalytic efficiency (kcat/Km) compared to the wild-type enzyme. Meanwhile, the stereoselectivities of these variants were improved from 43.0% ee of wild-type GoCR to >99% ee. In addition, mut-W193L/C93I/I187L displayed improved thermostability simultaneously. Theoretical structural analysis revealed that the changes in the catalytic pocket microenvironment resulted in the concurrent improvement of enzyme activity and thermostability. In the batch production of (R)-HPBE, up to 371 g L-1 substrate loading was completely reduced by utilizing the most efficient variant mut-W193L/C93I/I187L at 40 °C, affording (R)-HPBE with >99% ee and a space-time yield of 540.4 g L-1 per day. This study provides a potential and attractive biocatalyst for the efficient synthesis of (R)-HPBE.
Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis
Qu, Ge,Lonsdale, Richard,Yao, Peiyuan,Li, Guangyue,Liu, Beibei,Reetz, Manfred T.,Sun, Zhoutong
, p. 239 - 246 (2018/02/09)
Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building “small but smart” mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets. By using the synthetically difficult-to-reduce prochiral ketone tetrahydrofuran-3-one as a substrate, highly R- and S-selective variants were obtained (92–99 % ee) with minimal screening. The origin of stereoselectivity was provided by molecular dynamics analyses, which is discussed in terms of the Bürgi–Dunitz trajectory.
