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12.5 Conclusions 279

A nitrilase with a preference for fumaronitrile was also obtained by expressing
a gene sequenced in a photosynthetic cyanobacterium, Synechocystis sp. PCC6803
[12]. This enzyme, however, also hydrolyzed other substrates (aromatic, aliphatic,
dinitriles), albeit at reaction rates two orders of magnitude lower than fumaronitrile.

12.4.4
Cyanide-Transforming Enzymes
Cyanide hydratase activities were predicted for the putative proteins whose genes
were sequenced in the saprophytic fungi Aspergillus nidulans and N. crassa and in
the phytopathogenic fungus Gibberella zeae. Following the gene amplification from
genomic or cDNA and expression in E. coli, the expected activities were confirmed
and the enzymes compared to each other and to a previously characterized cyanide
hydratase from Gloeocercospora sorghi. Their specific activities, pH profiles, and
thermal stabilities were found to be different for each enzyme. The one from N.
crassa exhibited the best operational properties, and the ability to degrade KCN
from electroplating bath wastes with high silver or copper content [5].
Further, E. coli cells expressing the cyanide hydratases genes from A. niger
and P. chrysogenum were found to exhibit activities for both HCN and some
nitrile compounds, preferably 2-cyanopyridine (1–3.6% relative activity compared
to HCN) [6]. Dual nitrilase/cyanide hydratase activities were also described for the
enzymes in Fusarium oxysporum and Fusarium lateritium [36, 37]. It is possible that
this dual activity is a general feature of cyanide hydratases but has largely gone
unnoticed.

12.5
Conclusions

In this overview, the usefulness of genome mining for the search of new biocatalysts
was demonstrated by nitrilase studies. This approach is gaining in importance as the
wealth of bioinformatic data grows. The volume of unexploited nitrilase sequences
is still considerable. Recent sequential and structural analyses of wild-type and
mutant nitrilases and biochemical characterization of the enzymes has provided
a considerable amount of knowledge of their structure–activity relationships,
although their crystal structures are still largely unknown. This will enable a
rational design of further searches for new nitrilases meeting specific needs.

Acknowledgment

The support of the Czech Science Foundation, project P504-11-0394, Technol-
ogy Agency of the Czech Republic, project TA01021368, and internal project
RVO61388971 (Institute of Microbiology) is gratefully acknowledged.