The conversion of phenylglycinonitrile (2-aminophenylacetonitrile) by Escherichia coli strains was studied, which recombinantly expressed the arylacetonitrilase (NitA) from Pseudomonas fluorescens EBC191 and different nitrilase variants with altered reaction specificities. The whole-cell catalysts which formed the wild-type nitrilase converted (R,S)-phenylglycinonitrile preferentially to (S)-phenylglycine with a low degree of enantioselectivity. A recombinant strain which formed a variant of NitA produced mainly (S)-phenylglycine amide from (R,S)-phenylglycinonitrile and a second variant showed an almost complete enantioconversion and produced (R)-phenylglycine and left (S)-phenylglycinonitrile. The microbial-produced (S)-phenylglycinonitrile was used to study the chemical racemisation of (S)-phenylglycinonitrile at alkaline pH values in order to establish a dynamic kinetic resolution of the substrate. Subsequently, the conversion of (R,S)-phenylglycinonitrile by the whole-cell catalysts was studied at a pH of 10.8 which allowed a sufficient racemisation rate of phenylglycinonitrile. Surprisingly, under these conditions, strongly increased amounts of (S)-phenylglycine were formed by the recombinant E. coli cells expressing the amide-forming nitrilase variant. The aminopeptidase PepA from E. coli was identified by the construction of a deletion mutant and subsequent complementation as responsible amidase activity, which converted (S)-phenylglycine amide to (S)-phenylglycine.
Keywords: Amidase; Aminopeptidase; Nitrilase; Nitrile hydratase.