Mutagenesis of a fungal nitrilase from Gibberella intermedia for improved rate and different acid/amide

Engineering of a fungal nitrilase for improving catalytic activity and reducing by-product formation in the absence of structural information from Jin-Song Gong,   Heng Li,   Zhen-Ming Lu,   Xiao-Juan Zhang,   Qiang Zhang,   Jiang-Hong Yu,   Zhe-Min Zhou,   Jin-Song Shi and Zheng-Hong Xu

Catal. Sci. Technol., 2016, DOI: 10.1039/C5CY01535A

This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

Supporting a nitrile hydratase for better performance

There is a new accepted manuscript for the Journal of Molecular Catalysis B: Enzymatic which adds to the literature on the support of a nitrile hydratase to increase this notoriously sensitive enzyme’s robustness.  PVA/Chitosan-glutaraldehyde cross-linkednitrile hydratase as reusable biocatalyst for conversion of nitriles to amides by SV Pawar and GD Yadav  describes how to prepare a cross linked assembly containing the NHase from Rhodococcus rhodochrous ATCC BAA-870. When they test this immobilized enzyme in the hydration of 3-cyanopyridine, they find it is able to withstand a higher temperature and more basic conditions. They also show that their preparation is a reusable format which retains 50% of activity after 8 batches.

The figure below from the paper shows relative activity of free and immobilized NHase over time at 45 degrees C in 0.1M phosphate buffer. The upper line is the immobilized form.