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302 14 Enzymatic Stereoselective Synthesis of -Amino Acids

14.3.1.2 Nitrilase Substrate Selectivity
Many nitrilases have been screened for their ability to catalyze enantioselective
production of valuable carboxylic acid analogs with substitution in the α, β,or γ
position. For instance, nitrilase selectivity profiles identified a catalyst capable of
resolution of racemic mandelonitrile (an α-hydroxynitrile). Strains such as an Alcali-
genes sp. [44], Pseudomonas putida,and Microbacterium paraoxydans [45] showed
>93% ee when hydrolyzing mandelonitrile to (R)-mandelic acid (Table 14.1). In
another example, researchers at Diversa reported on the enantioselective conver-
sion of aromatic aminonitrile compounds for the production of α-amino acids [46].
The authors were successful in achieving a 79.5% yield of the (R)-acid product at
an enantiomeric excess of 96.3% from the substrate 4-fluorophenylglycinonitrile.
Recently we determined that two R. rhodochrous strains (A29 and A99) expressed
nitrilase activity after induction. These strains were capable of enantioselectively
hydrolyzing racemic 3-amino-3-phenylpropanenitrile directly to the corresponding
(R)-3-amino-3-phenylpropanoic acid with >95% ee (Table 14.1) in a kinetic resolu-
tion. Various inhibitors were used, that indicated the observed hydrolytic activity
was due to the presence of a nitrilase rather than a nitrile hydratase and amidase
pair [47].
Enantioselectivity at the γ position of alicyclic substrates has also been observed.
Winkler and colleagues [48] used commercial nitrilases (NIT 106 and NIT 107,
from Codexis, USA) to enantioselectively hydrolyze, conformationally constrained
γ-aminonitriles. Racemic five- and six-membered carbocylic γ-amino nitriles (both
cis and trans) were hydrolyzed enantioselectively to the corresponding amino acid
using the nitrilases. Cis isomers gave excellent enantiomeric excesses up to 99%,
while the reaction was less enantioselective for trans isomers (86% ee) (Table 14.1).

14.3.2
Nitrile Hydratase

Nitrile hydratases (EC 4.2.1.84) are metallo-enzymes that catalyze the hydration
of nitriles to their corresponding amides. From a structural viewpoint, nitrile
hydratases consist of two non-identical subunits (α and β), with similar molecular
masses of approximately 23 kDa. Nitrile hydratases exist as αβ dimers or α β
2 2
tetramers, with each αβ dimer binding a single metal atom. The amino acid
sequence of each subunit is unrelated, with the structural genes normally adjacent
to each other on the same operon [49]. Nitrile hydratases are classified into two
groups on the basis of their catalytic metal ion center: a nonheme iron atom [50] or
a noncorrinoid cobalt atom [51].
The distribution of nitrile hydratases in microorganisms is diverse, with bacteria
collected from shallow marine environments, varied geothermal locations, as well
as in numerous soil samples demonstrating this activity. Nitrile hydratases are
used as whole cell biocatalysts in industry to produce nicotinamide and acrylamide
[52].