Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor. Issue 2 (12th November 2019)

• Record Type: Journal Article
• Authors: Zhang, Qin; Lu, Xia‐Feng; Zhang, Yan; Tang, Xiao‐Ling; Zheng, Ren‐Chao; Zheng, Yu‐Guo
• Is Part Of: Biotechnology and bioengineering. Volume 117:Issue 2(2020)
• Page Start: 318
• Page End: 329
• Publication Date: 2019-11-12
• Subjects: nitrilase -- fragment swapping -- semi‐rational design -- enantioselectivity -- pregabalin precursor; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27203
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi‐rational design was developed for the engineering of nitrilase. A chimera Ba NIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine ( Aa NIT) and Brassica rapa ( Br NIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on Ba NIT to further generate variant Ba NIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4‐fold increase in whole‐cell activity and the enantiomeric ratio ( E ) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes. Abstract : Protein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions.