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13
Key-Study on the Kinetic Aspects of the In Situ NHase/AMase
Cascade System of M. imperiale Resting Cells for Nitrile
Bioconversion

Laura Cantarella, Fabrizia Pasquarelli, Agata Spera, Ludmila Mart´ ınkov´ a, and Maria
Cantarella

13.1
Introduction

The in situ nitrile hydratase/amidase (NHase/AMase) sequential enzymatic system
present in many microorganisms is a natural cascade system that has aroused
considerable interest in terms of its possible use in industrial bioprocesses − either
for the biotransformation of nitriles into valuable amides and/or acids or for
environmental protection, specifically for converting nitrile wastes into acids that
are less toxic and more easily degradable. So far, most application studies related
to the enzymatic hydrolysis of nitriles have made use of whole cells, which can
be easily prepared from culture collection strains at low cost [1]. In contrast to
metal catalysts, the hydration of nitriles catalyzed by NHase occurs under very
mild conditions, with high selectivity and yields, and avoiding waste production
[2, 3], thus representing a possible less expensive green process. Therefore, in
recent decades nitrile-converting enzymes have been increasingly used in organic
synthesis to develop more efficient and cleaner routes. Taking advantage of the
regio- and the stereoselectivity of the enzymatic system, a variety of racemic nitriles
were transformed into enantiopure amides or carboxylic acids [4–9].
The detoxification of organonitriles via bioprocesses is also expected to become a
cheaper process than the present chemical or physical methods [10–12]. Moreover,
important industrial achievements based on NHase-catalyzed bioprocesses have
been reported, such as the biotransformation of acrylonitrile into acrylamide,
of 3-cyanopyridine into nicotinamide, and of adiponitrile into 5-cyanovaleramide
[13, 14].
The NHase/AMase cascade enzymatic system transforms nitriles via atwo-step
reaction: the first one catalyzed by a NHase (EC 4.2.1.84), adds a water molecule
nitrile group, thus forming the corresponding amide, which is then transformed
into a carboxylic acid and ammonia in an AMase (EC 3.5.1.4) catalyzed reaction
[15–17]. This NHase/AMase cascade enzymatic system has been isolated and
characterized in various microorganisms, as reviewed in [1, 4, 15]. Numerous
kinetic aspects of these enzymes have also been elucidated and parameters such

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.