Conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191

Abstract

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5-6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.g. chloro- or hydroxy-groups) close to the nitrile group. Aliphatic dinitriles were attacked only at one of the nitrile groups and with most of the tested dinitriles the monocarboxylates were detected as major products. In contrast, fumarodinitrile was converted to the monocarboxylate and the monocarboxamide in a ratio of about 65:35. Significantly different relative amounts of the two products were observed with two nitrilase variants with altered reaction specifities. NitA converted some aliphatic substrates with higher rates than 2-phenylpropionitrile, which is one of the standard substrates for arylacetonitrilases. This indicated that the traditional classification of nitrilases as "arylacetonitrilases", "aromatic" or "aliphatic" nitrilases might require some corrections. This was also suggested by the construction of some variants of NitA which were modified in an amino acid residue which was previously suggested to be essential for the conversion of aliphatic substrates by a homologous nitrilase.

Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods

“…5). For example, we observed that Pseudomonas fluorescens NitA (Nit3 in our study) exhibited both arylacetonitrilase and aliphatic nitrilase activities, confirming previous reports 22 .…”

Genetic and Functional Diversity of Nitrilases in Agaricomycotina

“…The reaction of HCN was terminated by adding 1.0 mL of methanol and centrifugation. The activities for β-CA were calculated from the production of ammonia, which was determined spectrophotometrically [11,49]. The activities for HCN were determined from the production of formamide, which was determined spectrophotometrically [50] and by HPLC (see below).…”

“…The bacterial NLases were usually classified into a number of substrate-specificity subtypes, i.e., aromatic NLases, aliphatic NLases, arylacetoNLases, bromoxynil-specific NLases, CynHs and CynDs [7]. However, a recent study suggested that aliphatic NLases may not actually exist, since the activities for aliphatic nitriles are present in other NLase types such as arylacetoNLases [11]. The plant NLases could be classified into two main subtypes, namely NIT1 through NIT3 with broad substrate specificities on the one hand [12] and NIT4 with a strict specificity for β-cyanoalanine (β-CA) on the other [13].…”

Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase

“…The ''bienzymatic catalysts'' described in the present manuscript in combination with the previously constructed recombinant strains which simultaneously express the (S)-specific oxynitrilase from cassava together with the nitrilase(variants) allow the facile synthesis of a wide range of chiral hydroxycarboxylic acids and amides from simple precursors. This can be deduced from the ''proof of principle'' experiments described in this and previous publications for the ''bienzymatic catalysts'' and the substrate ranges of the used nitrilase and oxynitrilases which demonstrated for all three enzymes (the oxynitrilases from cassava and Arabidoposis thaliana and the nitrilase from P. fluorescens EBC 191) rather broad substrate specifities (Andexer et al 2007;Baum et al 2012;Brunner et al 2018;Bühler et al 2003;Kiziak et al 2005, Sosedov et al 2009.…”