Influence of point mutations near the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and Neurospora crassa

Arylacetonitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A (NitAn and NicNc, respectively) were expressed in recombinant Escherichia coli JM109. The respective whole-cell catalysts preferentially hydrolyzed (R)-enantiomers of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. NitNc also formed significant amounts of mandelamide from (R,S)-mandelonitrile (40% of total product), mainly found as the (S)-enantiomer. At pH 4.5 and 5.0, the cells retained more than 40 and 60% of their nitrilase activity at pH 7, respectively. Nitrilase variants generated by site-directed mutagenesis carried amino acid replacements in the vicinity of the catalytically active cysteine residues (C162 and C167 in NitAn and NitNc, respectively). The conversions of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile by the nitrilase variants were compared to the wild-type nitrilases in terms of activity, enantioselectivity, and acid/amide ratio of the products formed. Thus the W168A variant of NitNc was identified, which formed significantly increased amounts of mandelamide and 2-phenylpropionamide, and which demonstrated an almost complete inversion of enantioselectivity for the conversion of (R,S)-2-phenylpropionitrile (from R- to S-selectivity).

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► Fungal nitrilases hydrolyzed (R,S)-mandelonitrile with a high R-selectivity.
► The enzymes retained a high ratio of activity at pH 4.5–5.0.
► Mutations near the active center exerted similar effects in distantly related nitrilases.
► (S)-Mandelamide production was the highest in Neurospora crassa W168A variant.
► This variant exhibited an S- instead of R-selectivity for 2-phenylpropionitrile.