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14.3 Nitrile Hydrolysis Enzymes 305

trans-isomers (Table 14.2). Reactions performed using benzyl protected trans-
aminocyclohexanenitriles gave rise to the corresponding carboxylic acids with
a high enantiomeric excess (95–99%) for all three strains tested (Rhodococcus
equi A4, Rhodococcus erythropolis NCIMB 11540, and Rhodococcus sp. R312). The
enantiomeric excess of the remaining nitrile was found to be particularly high
in thecaseof R. equi A4, whereas the intermediate amide products exhibited
poor enantiomeric excess values [62]. This implies that a selective nitrile hydratase
was acting in R. equi A4, leaving the unreacted nitrile with a high enantiomeric
excess, and that an amidase with matching enantioselectivity was converting the
intermediate amide to enantiopure acid (Table 14.2). The high enantiomeric excess
values seen for the acid produced by all three strains, regardless of nitrile hydratase
selectivity, supports the presence of a highly enantioselective amidase in the three
organisms. When using a tosyl protecting group, good enantioselectivity was also
measured in the conversion of the amide to the acid, whereas nitrile hydratase
enantioselectivity was much poorer when using R. equi cells but enhanced for R.
erythropolis cells.
In contrast to these results, the five-membered trans-aminonitriles yielded trans-
amides of high enantiopurity, whereas the respective acid products had poor
enantiomeric excess values. The results support an enantioselective amidase and a
nonselective nitrile hydratase, particularly for the benzyl protected substrate, where
all the nitrile was converted [62].
Ma et al. [1] examined the effect of an N-protecting group on the conversion
of β-aminoalkanenitriles by a strain of Rhodococcus eryrthropolis AJ 270. Whole
cell reactions were performed using resting cells expressing both nitrile hydratase
and amidase activity. The nitrile in each case was converted to the intermediate
amide and the corresponding acid with opposite stereochemistry in >99.5% ee
when employing an N-benzyl protecting group (Table 14.2). In the absence of the
protecting group, the enantiomeric excess of the acid product fell to 20.8% [1].
Thus, the presence of the N-benzyl group resulted in a dramatic improvement
1
in enantioselectivity. Variation of the R group resulted in minor changes to the
observed selectivity (Table 14.2). The presence of an ethyl or an isopropyl group
1
at R resulted in improved enantioselectivity (>99.5% ee) compared to a methyl
substituent. A cyclopropyl group proved slightly inferior to the ethyl and isopropyl
groups (Table 14.2). Interestingly, the same improvement in enantioselectivity
was observed for O-benzylated β-hydroxynitriles. The excellent enantioselectivities
observed for the amide and acid products were attributed by the authors to a slightly
selective nitrile hydratase in combination with a highly enantioselective matching
amidase.
Our original studies with unprotected β-hydroxynitriles showed that these
compounds were hydrolyzed by R. rhodochrous ATCC BAA-870, expressing a
benzamide-induced cobalt type nitrile hydratase, to the corresponding amides and
acids [11]. The formation of the amide implies a nitrile hydratase and amidase
system (although sometimes nitrilases can release partially hydrolyzed substrates
as amides [63]). Further studies in our laboratories demonstrated that the system
was indeed a nitrile hydratase and amidase cascade reaction functioning via a two