Engineering of a nitrilase through consensus sequence analysis and conserved site substitution to improve its thermostability and activity

The thermostability of nitrilase from different microorganisms varies widely and is closely related to conserved sequences. In this study, a semi-rational design based on sequence analysis was performed to improve the thermostability and specific activity of the regioselective nitrilase from Acidovorax facilis ZJB09122. Using thermostable nitrilases from thermophilic microorganisms as templates, consensus sites 151, 223, 250, 169 and 280 were found to have effects on the thermostability and enzyme activity. A triple mutant AcN-T151V/C223A/ C250G (AcN-T) was obtained with 15.9% higher activity and 2.1-fold longer half-life at 50 degrees C compared with the parent AcN-M. The optimal reaction temperature was 55 degrees C and the product tolerance was also improved. It was found that site 250 close to the catalytic active center plays a key role in the thermostability of nitrilase, and this role is universal among homologous nitrilases. On the other hand, sites 151 and 223 mainly affect enzyme activity, and this role is not universal.

Automated summary

In this study, a semi-rational design based on sequence analysis was used to improve the thermostability and specific activity of a regioselective nitrilase. A triple mutant was obtained with higher enzyme activity and longer half-life, and specific sites were identified to play key roles in thermostability and enzyme activity.