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278 12 Mining Genomes for Nitrilases

However, a different approach was used to obtain arylacetonitrilases from fungi
such as A. niger, N. crassa, Aspergillus oryzae, Nectria haematococca,and Arthroderma
benhamiae. The selected genes were synthetically prepared and expressed in E. coli,
being optimized according to the codon bias in this host [7, 8, 31].
Hence, the set of well characterized arylacetonitrilases is comprised of over 10
enzymes; those from Alcaligenes [23, 25, 32] and P. putida [24] being very similar
or even identical. Because of their high enantioselectivity, they were used to design
processes for R-mandelic acid production from mandelonitrile concentrations of up
to 600 mM [33]. Fungal arylacetonitrilases from A. niger or N. crassa demonstrated
comparable enantioselectivities [8, 28] to these bacterial nitrilases. The enzyme
from L. aggregata was found to be useful for the selective hydrolysis of up to
300 mM o-chloromandelonitrile into (R)-o-chloromandelic acid (a building block of
the antiplatelet agent Clopidogrel ® ) [30].
The newly obtained nitrilases from B. japonicum [9–11] and B. xenovorans [9]
are evolutionarily distant from the aforementioned enzymes, differing from both
of these groups in their low to no enantioselectivities for mandelonitrile. These
enzymes, as well as the moderately enantioselective nitrilase from A. benhamiae
[8], may be suitable for the enantioretentive transformations of optically pure
(S)-mandelonitrile, as has already been demonstrated with the enzyme from P.
fluorescens [34].

12.4.2
Aromatic Nitrilases

The best characterized aromatic nitrilases were from rhodococci, but these enzymes
have also been known to occur in fungal strains [2]. New members of this
subgroup were obtained by searching fungal genomes [6]. The sources of these
enzymestobeexpressedin E. coli were Gibberella moniliformis, Penicillium marneffei,
Penicillium chrysogenum,and Meyerozyma guilliermondi. The enzymes were found
to act preferably on cyanopyridines like the bacterial enzymes but, in most cases,
also on acetonitrile, propionitrile, or valeronitrile. This is in accordance with the
substrate specificities of some nitrilases in the taxonomically related Fusarium
species (for a review see [3]) and in Gibberella intermedia [35].

12.4.3
Aliphatic Nitrilases

A nitrilase sequenced in the archaeon P. abyssi GE5 was expressed in E. coli to
give a highly thermostable enzyme (with an enzyme half-life of 25, 9, and 6 h
◦
at 70, 80, and 90 C, respectively [14]). This enzyme is the only experimentally
confirmed nitrilase to have been crystallized [13]. The low identity of this enzyme
to other characterized nitrilases makes its usefulness for homology modeling low.
This enzyme also differs from most other characterized nitrilases in its substrate
specificity, its preferential substrates being fumaronitrile and malononitrile [14].