Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid

Protein engineering of nitrilase for chemoenzymatic production of glycolic acid

“…However, work by others on the related enzyme cyanide dihydratase suggests that alteration of the quaternary structure imparted by a mutation may be one explanation Sewell et al, 2003). Protein engineering has also been used to increase the specific activity of a nitrilase from Arabidopsis thaliana >2.7-fold for the stereo-specific conversion of racemic isobutylsuccinonitrile to (3S)-3-cyano-5-methyl hexanoic acid (Xie et al, 2006). The two mutations F168V and L201N were combined to produce a nitrilase mutant that demonstrated specific activity 15.3-fold greater than the native enzyme, which suggests a synergistic effect on activity by these two mutations.…”

Improved Fitness of Arabidopsis thaliana Nitrilase 2

“…[8] Other characterized or recombinantly expressed plant nitrilases are known from Brassica rapa subspecies rapa, [9] Brassica rapa subspecies pekinensis, [10] Zea mays, Lupinus angustifolius, Nicotiana tabacum, Oryza sativa, and Brassica napus. [6,11] Especially the nitrilases from Brassica rapa [9] and Zea mays [12] are well characterized from the enzymatic point of view.…”

“…Whereas the substrate specificity for NIT4 is strongly restricted to b-cyano-l-alanine, [14] the three other isoenzymes show a similar substrate preference, accepting a variety of aromatic and aliphatic nitriles. [15] Besides several scientific literature reports about the selective hydrolysis of diverse nitriles using Arabidopsis thaliana nitrilases, [11,[16][17][18][19] there is also a patent application claiming their industrial potential. [20] The nitrilase I of A. thaliana was used in a directed evolution approach to improve its activity towards isobutylsuccinonitrile.…”

“…[20] The nitrilase I of A. thaliana was used in a directed evolution approach to improve its activity towards isobutylsuccinonitrile. [11] Several other literature reports exist which show the successful application of directed evolution as a tool for protein engineering. [21][22][23][24][25] The general advantage of nitrilases to perform hydrolysis under mild reaction conditions such as room temperature and neutral pH [26] is well known.…”

“…[28] The screening of 6700 variants revealed three hits (Table 1, entry 2-4) from the first round of ep PCR. A sequence alignment of Arabidopsis thaliana nitrilase NIT1 and NIT2 showed that the mutation of variant 1/7E (C230S) was identical to the mutation C236S from NIT1, which was created by ep PCR for improved activity towards isobutylsuccinonitrile by Xie et al [11] For further improvement of the activity, the mutations of the three best hits were combined by site directed mutagenesis. Thereby the activity was doubled compared to the individual mutants ( Devoid of detailed structural information, it is difficult to specify the influence of each mutation.…”