Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, β-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.

• Klíčová slova
• Agaricomycotina, Basidiomycota, cyanide hydratase, homology modeling, nitrilase, nitrile, overproduction, phylogenetic distribution, substrate docking, substrate specificity,
• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• Basidiomycota klasifikace   enzymologie   genetika   MeSH
• fumaráty metabolismus   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• kyanovodík metabolismus   MeSH
• pyridiny metabolismus   MeSH
• simulace molekulového dockingu MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminohydrolasy MeSH
• fumaráty MeSH
• fumaronitrile MeSH Prohlížeč
• fungální proteiny MeSH
• kyanovodík MeSH
• nitrilase MeSH Prohlížeč
• pyridiny MeSH

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

• Klíčová slova
• Aldoxime–nitrile pathway, Biocatalytic applications, Database mining, Heterologous production, Metagenome mining, Nitrilase,
• MeSH
• aminohydrolasy genetika   metabolismus   MeSH
• Bacteria enzymologie   genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biodegradace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• fungální proteiny genetika   metabolismus   MeSH
• houby enzymologie   genetika   MeSH
• metagenomika MeSH
• nitrily metabolismus   MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aminohydrolasy MeSH
• bakteriální proteiny MeSH
• fungální proteiny MeSH
• nitrilase MeSH Prohlížeč
• nitrily MeSH
• rekombinantní proteiny MeSH

The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.

• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• Arthrodermataceae enzymologie   MeSH
• Aspergillus niger enzymologie   MeSH
• druhová specificita MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• koncentrace vodíkových iontů MeSH
• kyseliny mandlové metabolismus   MeSH
• Nectria enzymologie   MeSH
• Neurospora crassa enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminohydrolasy MeSH
• fungální proteiny MeSH
• kyseliny mandlové MeSH
• mandelic acid MeSH Prohlížeč