Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles

Abstract

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.

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus

“…The NIT4 homologue (WP_014332611.1) from Sinorhizobium fredii , a nitrogen-assimilating bacterium, was recently purified by us and its activity for β-CA (≈2 U mg −1 protein) was determined. Other substrates of this enzyme were e.g., FN, PAN or various arylaliphatic dinitriles [ 17 ]. This protein exhibited ca.…”

“…Nitriles (substrates) and authentic standards of carboxylic acids and amides (reaction products) were from standard commercial sources except for (2 E )-3-cyanoacrylamide and (2 E )-3-cyanoacrylic acid—the products of transformation of FN, which were previously prepared by us [ 17 ].…”

“…Fungi, mainly Ascomycota , are also a source of cyanide hydratases (CynHs), whose specific activities are the highest for HCN. CynHs are related to NLases with below 40% amino acid sequence identities (hereinafter only identities) [ 15 , 16 , 17 ].…”

“…In Basidiomycota , the first three NLases and a CynH were recently described by us [ 17 , 18 ]. They were overproduced in E. coli and their activities were examined using whole cells [ 18 ] except for the arylacetoNLase from Auricularia delicata [ 17 ], which was purified. This enzyme is unique in Basidiomycota , having no close homologues in this division [ 18 ].…”

The use of clade‐specific PCR assays to identify novel nitrilase genes from environmental isolates

“…Due to these sequence variations, many techniques are employed in novel enzyme discovery, such as metagenomic library construction and functional screening (Robertson et al, 2004;Soares Bragance et al, 2017) whole genome sequencing, and data mining (Kaplan et al, 2011;Sharma & Bhalla, 2012;Veselá et al, 2016).…”