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References 293

the feed, without need of enzyme purification [34]. The influence of acrylonitrile
on AMase activity seemed obvious and effective. The bacterial transformations of
nitriles into amides are only documented for some industrial applications, in which,
apparently, amide hydrolysis was hampered by an unfavorable steric interaction
between the AMase and its substrate [1].
Feng et al. [35] also reported on the inhibition and/or inactivation of AMase in the
bi-sequential enzymatic system of Mesorhizobium sp. F28, a higher acrylamide yield
being obtained at a higher initial acrylonitrile concentration. The inhibitory effects
of high concentrations of acetonitrile and acetamide on NHase and AMase activity
from Rhodococcus pyridinivorans S85-2 and Brevundimonas diminuta AM10-C-1,
respectively, have also been reported [12].

13.5
Concluding Remarks

The reported investigations confirmed the different temperature dependence of the
two enzymes of the cascade system; the NHase could only operate in a narrow range
◦
of temperatures, while the AMase was stable up to 50–60 C. The NHase was very
sensitive to a high concentration of its substrate, and in some cases, an irreversible
inactivation occurred. The AMase activity was also affected by a combined effect
of temperature and high nitrile concentration. Whenever the process target is
the intermediate amide, the key to success would be the suppression and/or the
inactivation of AMase activity. The controlled inactivation of AMase in acrylonitrile
bioconversion made it possible to halt the reaction at the amide stage without
purifying NHase.
Based on these indications, in a real process, the integral bioreactor should be
optimized, and whenever the nitriles are able to inactivate the enzymes of the
cascade, the complete and rapid conversion of the substrate should be aimed for,
in order to minimize the inactivation of the enzyme.

Acknowledgments

The authors gratefully acknowledge the financial support from the University of
L’Aquila, and ESF COST Action CM0701 ‘‘CASCAT – Cascade Chemoenzymatic
Processes – New Synergies between Chemistry and Biochemistry’’. L. Mart´ ınkov´ a
would like to acknowledge the financial support from project P504/11/0394 (Czech
Science Foundation).

References
1. Mylerov´ a, V., and Martinkov´ a, L. 2. Linthorst, J.A. (2010) An overview:
(2003) Synthetic applications of nitrile- origins and development of green
converting enzymes. Curr. Org. Chem., chemistry. Found. Chem., 12,
7, 1–17. 55–68.