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
• Aminohydrolases chemistry   genetics   metabolism   MeSH
• Arthrodermataceae enzymology   MeSH
• Aspergillus niger enzymology   MeSH
• Species Specificity MeSH
• Fungal Proteins chemistry   genetics   metabolism   MeSH
• Phylogeny MeSH
• Hydrogen-Ion Concentration MeSH
• Mandelic Acids metabolism   MeSH
• Nectria enzymology   MeSH
• Neurospora crassa enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aminohydrolases MeSH
• Fungal Proteins MeSH
• Mandelic Acids MeSH
• mandelic acid MeSH Browser

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.

• MeSH
• Aminohydrolases chemistry   genetics   metabolism   MeSH
• Hydro-Lyases chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   MeSH
• Fungal Proteins chemistry   genetics   metabolism   MeSH
• Genome, Fungal * MeSH
• Genomics MeSH
• Fungi enzymology   genetics   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Sequence Analysis, DNA MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Alignment MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Aminohydrolases MeSH
• cyanide hydratase MeSH Browser
• Hydro-Lyases MeSH
• Fungal Proteins MeSH
• nitrilase MeSH Browser
• Recombinant Proteins MeSH