Organic Process Research & Development 2009, 13, 584–589
Rapid Production of Nitrilase Containing Silica Nanoparticles Offers an Effective
and Reusable Biocatalyst for Synthetic Nitrile Hydrolysis
Joshua D. Swartz,† Scott A. Miller,†,‡ and David Wright*,†
Vanderbilt UniVersity, Department of Chemistry, Station B 351822, NashVille, Tennessee 37235-1822, U.S.A., and UniVersity of
South Alabama, Department of Chemistry, 307 UniVersity BouleVard North, Mobile, Alabama 36688, U.S.A.
Abstract:
combination of nitrile hydratase and nitrile amidase enzymes
or by members of the nitrilase superfamily.12 There are at least
nine identified families within the nitrilase superfamily. One
well-studied gene cluster from the nitrilase superfamily is the
NIT1-3 cluster from Arabidopsis thaliana.13 These enzymes
utilize an active-site cysteine residue to catalyze the direct
conversion of aliphatic and aromatic nitriles into carboxylic
acids coupled with ammonia liberation and accept a wide array
of substrates. While nitrilase-containing cell farms have found
a foothold in the commercial production of some high-value
fine chemicals,14 they are not commonly employed in synthetic
strategies due to traditional barriers of enzymatic catalysis. In
general, enzymes are more expensive than many reagents, are
not easily recycled after use, and may have poor shelf lives if
they are not capable of activity following lyophilization or
freezing. These limitations have limited the large-scale industrial
use of nitrilase to maintaining various microbes expressing high
levels of nitrilase, such as with acrylamide production (∼6000
tons per year).15,16 Herein we report the encapsulation of a
cysteine active, recombinant nitrilase inside silica nanoparticles.
The reaction is performed in an aqueous buffer and mediated
by a water-soluble PAMAM dendrimer to yield a nitrilase-
containing silica nanocomposite capable of enzymatic conver-
sion of nitriles, easy separation from product, and reusability.
Rapid and efficient immobilization of nitrilase within silica nano-
particles overcomes many hurdles associated with biocatalysis. A
water-miscible dendrimer catalyzes the condensation of silicic acid
to silica dioxide, entrapping electrostatically bound nitrilase
molecules. Michaelis-Menten kinetics shows encapsulated nitrilase
functions similarly to free nitrilase in solution. Additionally, HPLC
analysis demonstrates that simple benchtop separation and recy-
cling of the biocatalyst over 10 individual reactions are achieved
without significant loss of enzyme and/or function. These findings
broaden the use of nitrilases in the production of fine chemicals
as well as general syntheses by overcoming some of the traditional
barriers associated with enzyme reagents and nitrile conversion.
Introduction
Nitrile compounds are simple aliphatic and aromatic me-
tabolites, cyanoglucosides, and cyanolipids serving as key
compounds and intermediates in a myriad of biochemical
pathways.1 The common biochemical transformation of orga-
nonitrile hydrolysis to higher value amide and carboxylic acid
groups is often inaccessible to organic synthetic strategies due
to the harsh conditions required for hydrolysis.2-5 Synthetically,
organonitriles are easily produced via the addition of cyanide
to alkyl halides, the Strecker reaction,6-9 reaction of aryl halides
with copper cyanide,10 and the dehydration of amides.11
However, the strong acids and bases, often in conjunction with
reflux conditions required to achieve nitrile hydrolysis, prove
impractical during the synthesis of sensitive and complex
molecules.
Results
In order to improve upon the implementation and recycling
of nitrilase for large-scale production and small-scale research
and development, we encased a commercially available nitrilase
within silica nanospheres using a biomimetic template. Previous
studies of oceanic diatoms have isolated a class of proteins,
called silaffins, that catalyze the condensation of silicic acid
into intricate exoskeletons.17 This process is amplified by
polycationic lysine modifications containing many secondary
and primary amines along the Sil-1A peptide.18 Subsequent
reports indicated that similar condensation chemistry could be
achieved using a variety of amine sources.19-23 Previously, we
Microbial organonitrile transformations are achieved bio-
chemically in ambient, aqueous environments by either a
* Author for correspondence. E-mail: David.Wright@Vanderbilt.edu. Fax:
615-343-1234. Telephone: 615-322-2636.
† Vanderbilt University.
‡ University of South Alabama.
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Vol. 13, No. 3, 2009 / Organic Process Research & Development
10.1021/op9000065 CCC: $40.75 2009 American Chemical Society
Published on Web 04/08/2009