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12.4 Enzyme Properties and Applications 277

As in wild-type enzymes bearing different residues, C-terminally adjacent to the
catalytic Cys (Ala vs Trp; see earlier text), the importance of this residue to nitrilase
enantioselectivity was observed in the enzyme variants [26]. The replacement of
A165 in NitP with G, F, Y, H, E, or R increased the enantioselectivity for (R)-
mandelic acid formation and altered the degree of amide formation. The impact
of the corresponding residue on these enzyme properties was also confirmed in
nitrilases from Alcaligenes faecalis (NitA [26]), N. crassa (NitNc), and Aspergillus niger
(NitAn) [28].
The N-terminally adjacent residue also affected enantioselectivity and amide
formation as documented by an examination of NitP variants (C163Q, C163N, etc.
[27]).
The effect of the residue three positions downstream of the catalytic Cys in
NitAn and NitNc was not unambiguous, although it seemed tempting to suggest
its importance for amide formation (Asn in cyanide hydratases, His in the majority
of nitrilases). Mandelamide formation increased in the H165N variant of NitAn,
but decreased in the corresponding variant of NitNc, H170N [28].

12.4
Enzyme Properties and Applications

The spectrum of known nitrilases and cyanide hydratases currently consists of a
significant part of enzymes obtained by genome mining. These enzymes were, in
many cases, helpful to the improvement of processes employing the hydrolysis of
industrially important nitriles.

12.4.1
Arylacetonitrilases

Arylacetonitrilases are attractive catalysts because of their enantioselective hydroly-
sis potential [29]. Much attention was focused on the investigation of enzymes that
were able to hydrolyze (R,S)-mandelonitrile into (R)-mandelic acid, which has a
number of applications (drug synthesis intermediate, resolving agent [24]). Several
such nitrilases were prepared by expressing in E. coli the genes from environmental
isolates (Alcaligenes, Pseudomonas [23–25]), or from metagenomes [4].
A number of new arylacetonitrilases were obtained by genome mining. Three
enzymes acting on, for example, mandelonitrile and phenylacetonitrile, were
prepared by expressing the genes from the sequenced genomes of B. japonicum
USDA110 (two enzymes [9–11]) and B. xenovorans LB400 [9] (see Section 9.3.2).
A similar approach was used to obtain enzyme(s) suitable for the enantioselective
hydrolysis of o-chloromandelonitrile. Six enzymes acting on this substrate were
encoded by the genes in Agrobacterium radiobacter, Sphingomonas wittichii, She-
wanella woodyi, Hoeflea phototrophica, Erwinia billingiae,and Labrenzia aggregata.
The highest activity of the crude enzyme was found in E. coli expressing the gene
from L. aggregata [30].