Page 282 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

258 11 Nitrile Converting Enzymes Involved in Natural and Synthetic Cascade Reactions

Nitrile
hydratase Amidase
RCN RCONH 2 RCOOH
0.24 M (NH 4 ) 2 SO 4
0.5 M NH 2 OH Amidase

RCONHOH

R = Ph, 3-MePh, 3-ClPh, CH 3 CH 2 , CH 3 (CH 2 ) 2 , CH 3 (CH 2 ) 3
Figure 11.10 Bienzymatic transformations of nitriles into the corresponding hydroxamic
acids [54].

high-value-added products (for a review, see [53]). Recently, this reaction was
coupled with a preceding NHase-mediated nitrile hydration step ([54]; Figure 11.10).
The NHase and amidase either originated from the same strain or different organ-
isms. The two reactions had to be carried out separately, as the high concentration
of hydroxylamine (e.g., 500 mM) used in the acyl transfer reaction inhibited the
NHase. However, the amide intermediate did not need to be isolated. When using
a crude cell extract with both NHase and amidase activity, the formation of the car-
boxylic acid in the first step was suppressed by the addition of high concentrations
of ammonium ions (e.g., 240 mM). This effect was probably caused by a transfer of
the acyl group from the donor to ammonia as the acceptor (a stronger nucleophile
than water), leading to the formation of an amide. Alternatively, a recombinant
NHase in a strain that contains no amidase activity could be employed in the first
step. This cascade reaction could also be used to determine the activity of NHases,
by transforming the amides into hydroxamic acids, which can be easily determined
spectrophotometrically.

11.3.2
Nitrilase–Amidase

Nitrilase is a useful alternative to the NHase/amidase cascade, the benefits of
nitrilase often being its higher enantioselectivity or stability. However, a drawback
of nitrilase is its tendency to form mixtures of carboxylic acids and amides, this
phenomenon being caused by an untypical cleavage of the reaction intermediate
leading to the amide as the side product [55].
Therefore, in order to obtain the carboxylic acid as the final product, it was
proposed to transform the amide using an amidase. The reactions can proceed in
a single reactor or in two reactors connected in series. The utility of this method
was exemplified by the hydrolysis of 4-cyanopyridine into isonicotinic acid [56],
an intermediate in the synthesis of the tuberculostatic drug isoniazid. Nitrilases
from Aspergillus niger or Fusarium solani and an amidase from R. erythropolis were
chosen for this process, as they exhibited compatible substrate specificities. If the
cascade reactions were carried out in two separate reactors, a crude extract from
R. erythropolis A4 cells could serve as the amidase source. In a one-pot reaction,
the NHase present in this extract would compete with the nitrilase and increase