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Abstract 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.

The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.

To examine nitrilase-mediated hydrolysis of nitriles to produce optically pure α-hydroxycarboxylic acids.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 max and K m of GPnor51 towards o-chloromandelonitrile were 1.9 μmol min(-1) mg(-1) protein and 0.38 mM, respectively. GPnor51 also demonstrated high enantioselectivity toward mandelonitrile and other substituted mandelonitrile.This enzyme has a great potential for commercial production of optically pure (R)-mandelic acid and its derivatives.

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−1 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−1 culture, respectively, with phenylacetonitrile as substrate.