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Nitrilases are the enzymes that catalyze the hydrolysis of nitriles to corresponding carboxylic acid and ammonia. They are broadly categorized into aromatic, aliphatic, and arylacetonitrilases based on their substrate specificity. Most of the studies pertaining to these enzymes in the literature have focused on aromatic and aliphatic nitrilases. However, arylacetonitrilases have attracted the attention of academia and industry in the last several years due to their aryl specificity and enantioselectivity. They have emerged as interesting biocatalytic tools in green chemistry to synthesize useful aryl acids such as mandelic acid and derivatives of phenylacetic acid. The aim of the present review is to collate information on the arylacetonitrilases and their catalytic properties including enantioselectivity and potential applications in organic synthesis. [ABSTRACT FROM AUTHOR]

Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided by combining nitrile and NADH conversion in a single reaction. This allowed the direct measurement of nitrile degradation and provided not only insights into substrate spectrum and specificity but also in degradation efficiency. The novel assay was used for investigation of candidate nitrilase-encoding genes. Seventy putative nitrilase-encoding gene and the corresponding deduced protein sequences identified during sequence-based screens of metagenomes derived from nitrile-treated microbial communities were analyzed. Subsequently, the assay was applied to 13 selected candidate genes and proteins. Six of the generated corresponding Escherichia coli clones produced nitrilases that showed activity and one unusual nitrilase was purified and analyzed. The activity of the novel arylacetonitrilase Nit09 exhibited a broad pH range and a high long-term stability. The enzyme showed high activity for arylacetonitriles with a KM of 1.29 mM and a Vmax of 13.85 U/mg protein for phenylacetonitrile. In conclusion, we provided a setup for simple and rapid analysis of putative nitrilase-encoding genes from sequence to function. The suitability was demonstrated by identification, isolation, and characterization of the arylacetonitrilase. Key points: • A simple and fast high-throughput nitrilase screening was developed. • A set of putative nitrilases was successfully screened with the assay. • A novel arylacetonitrilase was identified, purified, and characterized in detail. [ABSTRACT FROM AUTHOR]

Arylacetonitrilases have been widely acknowledged as important alternatives to chemical catalysts for synthesizing optically pure 2-hydroxyphenylacetic acids from nitriles. In this work, two residues (Thr132 and Ser190) located at the catalytic tunnel in the active site of an arylacetonitrilase nitA from uncultured organisms were mutated separately by site-directed mutagenesis. Ser190 was demonstrated to be the critical position which has a greater influence on arylacetonitrilase nitA activity than Thr132. The replacement of serine at position 190 with glycine increases its activity toward mandelonitrile and ( o, m, p)-chloromandelonitrile, whereas replacing it with leucine abolished its activity. The best mutant S190G exhibited threefold higher specific activity toward mandelonitrile compared with that of wild-type nitA, which rendered it promising for industrial application. Homology modeling and molecular docking experiments were in agreement with the kinetic assays and support the improved catalytic performance. [ABSTRACT FROM AUTHOR]

Objective: To examine nitrilase-mediated hydrolysis of nitriles to produce optically pure α-hydroxycarboxylic acids. Results: A novel nitrilase, GPnor51, from Luminiphilus syltensis NOR5-1B was discovered by genomic data mining. It could hydrolyze racemic o-chloromandelonitrile to ( R)- o-chloromandelic acid with high enantioselectivity ( ee 98.2 %). GPnor51 was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties studied. GPnor51 had a broad substrate acceptance toward various nitriles with structure diversity. It was an arylacetonitrilase that uses arylacetonitriles as optimal substrates. The V and K of GPnor51 towards o-chloromandelonitrile were 1.9 μmol min mg protein and 0.38 mM, respectively. GPnor51 also demonstrated high enantioselectivity toward mandelonitrile and other substituted mandelonitrile. Conclusion: This enzyme has a great potential for commercial production of optically pure ( R)-mandelic acid and its derivatives. [ABSTRACT FROM AUTHOR]

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity ( V) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition ( I) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg, Ag, Pb, and Co were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80-94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg dcw, which is highest in comparison to till reported organism. Arylacetonitrilase -mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.) [ABSTRACT FROM AUTHOR]

Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for ( R, S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine. [ABSTRACT FROM AUTHOR]

Purpose of work: Our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l culture, respectively, with phenylacetonitrile as substrate. [ABSTRACT FROM AUTHOR]

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH4)2SO4 fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant ( k cat/ K m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of ( R)-(−)-mandelic acid from readily available mandelonitrile. [ABSTRACT FROM AUTHOR]

A novel nitrilase which catalyzes the direct hydrolysis of arylacetonitrile derivatives to the corresponding arylacetic acids has been found in strain JM3, tentatively identified as Alcaligenes faecalis. The addition of isovaleronitrile greatly enhanced the formation of the nitrilase. The culture conditions for the best production of the nitrilase and the substrate specificity of the enzyme are described. [ABSTRACT FROM AUTHOR]