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.
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