Probing the enantioselectivity of a diverse group of purified cobalt-centred nitrile hydratases

Abstract

In this study a diverse range of purified cobalt containing nitrile hydratases (NHases, EC 4.2.1.84) from Rhodopseudomonas palustris HaA2 (HaA2), Rhodopseudomonas palustris CGA009 (009), Sinorhizobium meliloti 1021 (1021), and Nitriliruptor alkaliphilus (iso2), were screened for the first time for their enantioselectivity towards a broad range of chiral nitriles. Enantiomeric ratios of >100 were found for the NHases from HaA2 and CGA009 on 2-phenylpropionitrile. In contrast, the Fe-containing NHase from the well-characterized Rhodococcus erythropolis AJ270 (AJ270) was practically aselective with a range of different α-phenylacetonitriles. In general, at least one bulky group in close proximity to the α-position of the chiral nitriles seemed to be necessary for enantioselectivity with all NHases tested. Nitrile groups attached to a quaternary carbon atom were only reluctantly accepted and showed no selectivity. Enantiomeric ratios of 80 and >100 for AJ270 and iso2, respectively, were found for the pharmaceutical intermediate naproxennitrile, and 3-(1-cyanoethyl)benzoic acid was hydrated to the corresponding amide by iso2 with an enantiomeric ratio of >100.

Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids: Cascade reactions employing a hydroxynitrile lyase, nitrile-converting enzymes and an amidase

“…6) depends on the enantiomeric bias of the HnL and, hence, does not require an enantioselective NHase. This is important, because enantioselectivity in NHases, although much more common than once surmised [47], is still in an early phase of development. NHases are industrially relevant and to an increasing degree available from recombinant expression.…”

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases

“…The Klebsiella oxytoca NHase (KoNHase) [8] showed imbalanced overexpression with significant amounts of the β-subunit but almost no α-subunit. Nitriliruptor alkaliphilus NHase (NaNHase) [15] predominantly formed inclusion bodies and was detected in the insoluble fraction. A putative NHase from Rhizobium leguminosarum bv.…”

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

“…Thus, 2.8 g of atrolactamide (70% yield) was obtained. 1 2-Methyl-2-phenylpropionitrile and 2-methyl-2-phenylpropionic acid were purchased from Accela ChemBio Inc. (San Diego, CA), and 2-acetyloxy-2-methylphenylacetonitrile was kindly supplied by S. van Pelt, TU Delft (47). The sources of all other chemicals were described previously (41).…”