Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor
Xia‐Feng Lu, Hongjuan Diao, Zhe‐Ming Wu, Zilong Zhang, Ren‐Chao Zheng, Yu‐Guo Zheng
Abstract
Abstract Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase ( Ba NIT M0 ) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor ( S )‐3‐cyano‐5‐methylhexanoic acid (( S )‐CMHA). However, Ba NIT M0 also catalyzed the hydration of IBSN to produce by‐product ( S )‐3‐cyano‐5‐methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of Ba NIT M0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S ( Ba NIT M2 ) displayed higher enantioselectivity ( E = 515), increased enzyme activity (5.4‐fold) and reduced amide formation (from 15.8% to 1.9%) compared with Ba NIT M0 . Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio‐ and regio‐selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring Ba NIT M2 as biocatalyst afforded ( S )‐CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of Ba NIT M2 for efficient manufacturing of pregabalin.