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12
Mining Genomes for Nitrilases

Ludmila Mart´ ınkov´ a

12.1
Strategies in Nitrilase Search

Over the past, about 30 years, more than 100 studies have been published on the
use of nitrilases (EC 3.5.5.x) as catalysts for the hydrolysis of nitriles, the focus being
on enzymes suitable for the synthesis of industrially important products such as
mandelic acid and other hydroxy acids, mandelamide or unnatural amino acids,
often enantiopure compounds. In these works, several strategies were applied in
order to get new nitrilases (Figure 12.1).
The search for nitrilases was initially based on the selection of cultivable soil
bacteria using nitriles as substrates for their growth [1]. The nitrilases from some
of these isolates were purified and characterized, their amino acid sequences being
partially determined. This enabled the cloning, sequencing, and heterologous
expression of those nitrilase genes. Pioneering studies of this type were performed
with the nitrilase of Rhodococcus rhodochrous J1 and followed by structural studies
of this enzyme [2]. However, the purification of the enzymes from cultivable
isolates, or from the heterologous hosts carrying the genes amplified from them,
only enabled the acquisition of slightly over 20 enzymes by 2003 [3], and only
a few of them originated from plants or fungi. Although this strategy was not
completely abandoned in the last decade, other approaches such as the exploration
of metagenomes and databases have gained increasing importance. The spectrum
of known nitrilases was substantially broadened as a result of a study which
described, in terms of substrate specificities and enantioselectivities, a total of 137
nitrilases expressed from metagenomic DNA samples [4].
In the last decade, knowledge of the amino acid sequences of the characterized
nitrilases, along with an increasing number of sequenced genomes, made it possible
to search databases for new nitrilases. The genes were then either amplified from
the source organisms or prepared synthetically. By using the latter approach, the use
of the wild-type organisms (some of them pathogenic or difficult to cultivate) was
avoided, and, furthermore, the codon frequency of the genes could be optimized for
expression in Escherichia coli. Database mining made it possible to obtain nitrilases

Cascade Biocatalysis: Integrating Stereoselective and Environmentally Friendly Reactions, First Edition.
Edited by Sergio Riva and Wolf-Dieter Fessner.
c 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.