Page 331 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

14.3 Nitrile Hydrolysis Enzymes 307

enzyme system. The first reaction step, as catalyzed by nitrile hydratase, was
rapid but not enantioselective for β-hydroxy substituted phenoxy compounds. The
subsequent hydrolysis of the resultant amide to the corresponding carboxylic acid
by the amidase was considerably slower, but enantioselective (99% ee). The amidase
enantioselectivity was reduced when other β-substituted substrates were used, such
as 3-hydroxy-3-phenylpropanenitrile or 3-hydroxy-3-arylpropanenitrile [11].
In a subsequent study, we turned our attention to β-aminonitriles. Various aryl
substituted unprotected β-aminonitriles (3-amino-3-arylpropanenitriles) were syn-
thesized and enantioselective hydrolysis of these compounds to the corresponding
amides was achieved using R. rhodochrous ATCC BAA-870 [12]. Interestingly, initial
biocatalytic experiments did not result in the formation of hydrolysis products of
3-amino-3-phenylpropanenitrile. Further investigations proved that the charge on
the amine in the β-position made this compound a less suitable substrate than
the corresponding β-hydroxy compounds [11, 12]. This problem was overcome by
shifting the reaction pH higher, where a higher percentage of the amino groups
would be unprotonated. In order to avoid rapid denaturation of the enzyme at high
pH, a functional balance was achieved at pH 9 [12].
Unlike the results obtained for the β-hydroxy compounds, the main product
from the β-aminonitriles was determined to be the amide, rather than the acid
(Table 14.2). In fact, acid formation was only detected after extended incubation
times, indicating that the amidase responsible required some form of induction
or derepression that was not observed in the β-hydroxy studies. At this point, we
cannot be sure that the amidases responsible are the same for the β-hydroxy- and the
β-hydroxynitrile compounds. The accumulation of amide may indicate that there
is a mismatch between the optimal substrate profiles of the nitrile hydratases and
the amidases available in Rhodococci. Suitably matching amidases may, however,
be available from other microorganisms. Heck and coworkers [64] discovered β-
aminopeptidases from Sphingosinicella sp. capable of enantioselectively hydrolyzing
3
3
β -amino amides to the corresponding l-β -amino acids in greater than 99% ee.
Another difference between the catalysis of the β-amino and the β-hydroxynitrile
compounds is that the nitrile hydratase appears to have been partially enantios-
elective toward the β-aminonitriles, resulting in moderate enantiomeric excess
values for both the residual (R)-enantiomer of the parent nitrile and for the
(S)-enantiomer of the amide product, while the nitrile hydratase seemed non-
enantioselective when acting on β-hydroxynitrile compounds [11, 12]. Based on the
results of genome sequencing it appears that there is only one nitrile hydratase
present in R. rhodochrous ATCC BAA-870, and although a gene coding for a nitrilase
enzyme is present on the genome, no nitrilase activity was detected [65]. Hence,
the observed enantioselectivity seems to be substrate dependent.
Of interest is the fact that β-lactamases from Rhodococcus globerulus could be
used by Lloyd [66] to hydrolyze enantiomers of β-lactams to yield cyclic β-amino
acids with greater than 90% ee. β-Lactamases belong to the β-lactam-recognizing
enzymes (BLREs) superfamily. Their active site is characterized by a Ser-Ser-Lys
catalytic triad and an oxyanion hole [67], similar to amidase signature enzymes.