Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

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 BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on BaNIT to further generate variant BaNIT/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.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor

“…The enantiomeric excess (ee) of IBSN and CMHA were determined by chiral GC as described previously (Zhang et al, 2020).…”

“…In our previous work, a robust nitrilase Ba NIT/L223Q/H263D/Q279E ( Ba NIT M0 ) was developed by fragment swapping and semi‐rational design, which catalyzed asymmetric hydrolysis of high concentration of IBSN (100 g/L) with excellent enantioselectivity (Zhang et al, 2020). However, it was found that Ba NIT M0 exhibited poor reaction specificity towards IBSN.…”

“…However, it was found that Ba NIT M0 exhibited poor reaction specificity towards IBSN. Except for the hydrolysis activity, the nitrilase also catalyzed hydration of IBSN to produce the by‐product 3‐cyano‐5‐methylhexanoic amide (CMHM) (Zhang et al, 2020; Zhang, Wu et al, 2019). Occurrence of the hydration activity severely plagued application of the nitrilase in chemoenzymatic synthesis of pregabalin with high yield and purity.…”

Purification and Characterization of Nitphym, a Robust Thermostable Nitrilase From Paraburkholderia phymatum

“…Despite all the characterized nitrilases, no common feature of protein sequences was found that could explain their stability, only highly specific motifs were proposed as being important, thus making protein engineering complicated. Nevertheless, some nice examples have been reported to overcome stability limitations of nitrilases ( Xu et al, 2018 ; Zhang et al, 2020 ) in addition to other engineering work targeting enantioselectivity/specificity or substrate inhibition at high loadings ( Stolz et al, 2019 ).…”

Improving the activity and thermostability of GH2 β‐glucuronidases via domain reassembly

“…Enzymes, as biocatalysts, have drawn considerable attention in many fields due to their advantages over conventional catalysts, such as their mild reaction conditions and high selectivity (Bornscheuer et al, 2012; Rudroff et al, 2018; Wu et al, 2020). However, many enzymes have drawbacks, such as low expression levels, poor activity, and low stability, which have severely limited their industrial applications (Zhang et al, 2020). Protein engineering effectively solves these inherent problems of enzymes (Bornscheuer, 2016; Chen & Arnold, 2020; Whitehead et al, 2020).…”