Acrylamide production using encapsulated nitrile hydratase from Pseudonocardia thermophila in a sol-gel matrix

Article abstract of DOI:10.1016/j.molcatb.2013.11.014

The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) was successfully encapsulated in tetramethyl orthosilicate sol-gel matrices to produce a PtNHase:sol-gel biomaterial. The PtNHase:sol-gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min at 35 C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50% of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to acrylamide. The thermostability and long-term storage of the PtNHase:sol-gel are substantially improved compared to the soluble NHase. Additionally, the biomaterial is significantly more stable at high concentrations of methanol (50% and 70%, v/v) as a co-solvent for the hydration of acrylonitrile than native PtNHase. These data indicate that PtNHase:sol-gel biomaterials can be used to develop new synthetic avenues involving nitriles as starting materials given that the conversion of the nitrile moiety to the corresponding amide occurs under mild temperature and pH conditions.

Full text of DOI:10.1016/j.molcatb.2013.11.014

Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Acrylamide production using encapsulated nitrile hydratase from
Pseudonocardia thermophila in a sol–gel matrix
a
a
b
a,c,∗
Salette Martinez , Misty L. Kuhn , James T. Russell , Richard C. Holz
,
b,∗∗
Timothy E. Elgren
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL 60660, USA
Department of Chemistry, Hamilton College, Clinton, NY 13323, USA
Department of Chemistry, Marquette University, Milwaukee, WI 53201, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2013
Received in revised form
The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) was successfully
encapsulated in tetramethyl orthosilicate sol–gel matrices to produce a PtNHase:sol–gel biomaterial. The
PtNHase:sol–gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min
1
4 November 2013
◦
at 35 C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic
Accepted 18 November 2013
activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50%
of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to
acrylamide. The thermostability and long-term storage of the PtNHase:sol–gel are substantially improved
compared to the soluble NHase. Additionally, the biomaterial is significantly more stable at high concen-
trations of methanol (50% and 70%, v/v) as a co-solvent for the hydration of acrylonitrile than native
PtNHase. These data indicate that PtNHase:sol–gel biomaterials can be used to develop new synthetic
avenues involving nitriles as starting materials given that the conversion of the nitrile moiety to the
corresponding amide occurs under mild temperature and pH conditions.
Available online 1 December 2013
Keywords:
Nitrile hydratase
Sol–gels
Enzyme immobilization
Biomaterials
Cobalt
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
costs [2,4]. There are two metabolic pathways found in microor-
ganisms for nitrile degradation (Scheme 1). In the first pathway, the
nitrile is directly hydrolyzed to their corresponding carboxylic acid
and ammonia by nitrilase (EC 3.5.5.1) [3]. In the second pathway,
nitriles are first hydrated to their corresponding amide by nitrile
hydratase (EC 4.2.1.84), then the amide is subsequently hydrolyzed
to carboxylic acid and ammonia by amidase (EC 3.5.1.4) [3].
Microbial nitrile hydratase (NHase) has great potential as a bio-
catalyst for organic chemical processing because of its ability to
convert nitriles to amides under physiological conditions. How-
ever a major issue in the use of enzymes, in general, and NHases
specifically in organic synthetic processes is the difficulty in sep-
arating the enzyme from the synthetic reaction mixture [7]. A
related issue involves the use of aprotic solvents in organic syn-
thetic reaction mixtures, which renders most enzymes including
NHase inactive [7–9]. One way to overcome both of these hur-
dles is through the encapsulation of enzymes within silica glasses
derived through sol–gel processing [10–13]. Encapsulated enzymes
have resulted in the generation of novel functional materials
that are optically transparent and sufficiently porous to per-
mit small substrates access to the entrapped enzyme [11,14–19].
Recent studies have demonstrated that encapsulated proteins
retain their solution structure and native function while resid-
ing in the hydrated pore of the sol–gel [11,20–22]. Moreover,
Nitriles are used extensively to produce a broad number of spe-
cialty chemicals containing amines, amides, amidines, carboxylic
acids, esters, aldehydes, ketones, and heterocyclic compounds [1,2].
These compounds are used in a wide array of reactions as chemical
feedstocks for the production of solvents, extractants, pharma-
ceuticals, drug intermediates, pesticides (dichlobenil, bromoxynil,
ioxynil, buctril), and polymers [1]. For example, acrylonitrile and
adiponitrile are used in the production of polyacrylamide and
nylon-66, respectively, the latter of which is one of the most
important industrial polyamides derived from petroleum feed-
stocks [3–6]. However, the harsh industrial conditions needed to
hydrate nitriles to their corresponding amides (either acid or base
hydrolysis), are often incompatible with the sensitive structures
of many industrially and synthetically relevant compounds, which
decreases product yields and consequently increases production
∗ Corresponding author at: Department of Chemistry, Marquette University, Mil-
waukee, WI 53201, USA. Tel.: +1 414 288 7230.
∗
∗ Corresponding author. Tel.: +1 315 859 4695; fax: +1 315 859 4807.
E-mail addresses: Richard.holz@marquette.edu (R.C. Holz),
TElgren@Hamilton.edu (T.E. Elgren).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.11.014

2
0
S. Martinez et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
O
O
Nitrile hydratase
H O
Amidase
R
N
+
NH3
R
NH2
R
OH
2
H O
2
Nitrilase
H O
2
Scheme 1.
nanoscopic confinement within sol–gels stabilizes proteins against
thermal and proteolytic degradation [11,17]. These physical prop-
erties permit the broad application of sol–gel:protein materials as
chemical sensors, separation media, and heterogeneous catalysts
Subsequently, the PtNHase activator gene was inserted between
the NdeI and HindIII restriction sites of the ampicillin resistant
pET21a+ vector to create the Pt-activator/pET21a+ plasmid. All
plasmid sequences were confirmed using the automated DNA
sequencing at the University of Chicago Cancer Research Center
DNA sequencing facility.
[
23].
For industrial applications (i.e. acrylamide production) whole
cells containing NHase and some isolated, purified, NHases have
been immobilized on various supports by means of adsorption,
entrapment, cross-linking, and membrane immobilization [4].
While the immobilization of whole cells containing NHase have
been successful for the production of various commodity chem-
icals, purified NHases are necessary for the specific hydration of
nitriles with other hydrolysable groups that will be susceptible to
side-reactions within a bacterial cell. In addition, reaction systems
that cannot tolerate carboxylate side products also require puri-
fied NHase since other enzymes in the bacterial nitrile degradation
pathway, such as nitrilases can convert nitriles to carboxylates [24].
Purified enzymes also eliminate the need to have nitrile substrates
pass across cell membranes; this would otherwise decrease the
yields of recoverable products [4,25]. Herein we report the immo-
bilization of the Co-type NHase from Pseudonocardia thermophila
JCM 3095 (PtNHase) using the sol–gel process in tetramethyl ortho-
silicate (TMOS) gels. This novel biocatalytic material is capable of
specifically hydrating acrylonitrile to acrylamide in high yields and
under mild conditions.
2
.3. Expression and purification of recombinant PtNHase
The Pt-His /pET28a+ and Pt-activator/pET21a plasmids were
+
6
co-transformed into E. coli BL21(DE3) competent cells (Stratagene)
for expression. A 100 mL LB-Miller starter culture was inoculated
from a single colony with 50 ␮g/mL and 100 ␮g/mL of kanamycin
and ampicillin, respectively. A 6 L culture was inoculated from
this starter culture using 7 mL per liter and grown at 37 C until
an OD6
◦
of 0.6–0.8 was reached. The culture was cooled to
0 C, induced with 0.1 mM isopropyl ␤-d-1-thiogalactopyranoside
00 nm
◦
2
(IPTG), supplemented with 0.25 mM CoCl2 [28,29], and expressed
at the same temperature for 16–18 h. Cells were harvested by cen-
◦
trifugation at 6370 × g 4 C, for 5 min. The cells were resuspended
at 3 mL per gram of buffer A (50 mM NaH PO , pH 7.5, 500 mM
2
4
NaCl, 10 mM imidazole) then lysed by sonication on ice for 8 min
(30 s on, 45 s off) using a 21W Misonex sonicator 3000. Cell debris
◦
was removed by two rounds of centrifugation at 31,000 × g, 4 C,
for 20 min. The protein was purified by immobilized metal affin-
ity chromatography (IMAC) using Ni-NTA (nickel-nitrilotriacetic
acid) Superflow Cartridges (Qiagen). The column was equilibrated
with buffer A and the crude extract was loaded onto the column.
Unbound protein was washed with 15 column volumes (CV) of
buffer A with 25 mM imidazole. The protein was eluted with a lin-
ear gradient (0–100%) of buffer B (buffer A with 500 mM imidazole)
over 20 CVs at a flow rate of 2 mL/min. Peak fractions were pooled,
resuspended in buffer C (50 mM Tris–HCl pH 7.5, 300 mM NaCl) and
concentrated with an Amicon Ultra-15 centrifugal filter device with
a molecular weight cutoff (MWCO) of 30,000 (Millipore). The purity
of PtNHase was analyzed by using 12.5% SDS-PAGE. The protein
concentration was determined by UV absorbance at 280 nm using
2
. Experimental
2.1. Materials
Tetramethyl orthosilicate (TMOS, ≥99%), acrylonitrile,
acrylamide, Type I Trypsin from bovine pancreas, and Type
II ␣-chymotrypsin from bovine pancreas, were purchased
from Sigma–Aldrich. All reagents were of the highest purity
available and used as received without further purifica-
tion.
2.2. Plasmid construction
−
1
−1
a calculated molar extinction coefficient of 174, 640 M cm
A pUC18-NHase plasmid encoding the ␣-subunit and ␤-subunit
genes of PtNHase [26,27], was used as a template for the poly-
and the Coomassie (Bradford) Protein Assay Kit (Thermo Scien-
tific Pierce). The calculated molecular mass of the heterotetramer
was 101 kDa. Theoretical molecular mass and protein extinction
coefficients were calculated with the ExPASy ProtParam tool [30].
merase chain reaction (PCR). A polyhistidine (His ) affinity tag
6
was engineered onto the C-terminus of the ␣-subunit gene using
Phusion DNA polymerase. The following primers were used for
ꢀ
the PCR: forward 5 -GCC ATG GGA AAC GGC GTG TAC GAC GTC
ꢀ
ꢀ
GGC GG-3 and reverse 5 -GGT ACC AAG CTT TCA ATG ATG
ꢀ
2.4. Kinetic characterization of PtNHase
ATG ATG ATG ATG CGC GAC CGC CTT-3 . The PCR product (␤␣-
His6 genes) was subcloned into the pSC-B-amp/kan vector using
the Strataclone Blunt PCR cloning kit. The ␤␣-His6 genes were
subsequently subcloned between the NcoI and HindIII restric-
The activity of purified PtNHase was determined by measur-
ing the hydration of 100 mM acrylonitrile to acrylamide (225 nm,
−
1
−1
◦
+
ε = 2.9 mM cm ) in 50 mM Tris–HCl, pH 7.5 at 35 C. Assays were
performed in a 1 mL quartz cuvette in triplicate on a Shimadzu
UV-2450 PC spectrophotometer equipped with a TCC temperature
controller. One unit of enzyme activity was defined as the amount
of enzyme that catalyzed the production of 1 ␮mol of acrylamide
tion sites of the kanamycin resistant pET28a to create the
+
Pt-His /pET28a plasmid. The nucleotide sequence for the PtNHase
6
activator gene was obtained from the GeneBank, ID HV233497.1,
and used to synthesize the gene with E. coli codon usage and
cloned into a pIDT-SMART (Integrated DNA Technologies, Inc.)
ampicillin resistant vector with NdeI and HindIII restriction sites.
◦
per minute at 35 C.

S. Martinez et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
21
2.5. Encapsulation of PtNHase in tetramethyl orthosilicate
The sol consisted of 1.57 mL of TMOS, 0.350 mL Nanopure (18 ꢀ)
H O, and 0.011 mL of 0.040 M HCl. The mixture was sonicated
2
on ice for 30 min, and then left on ice for ∼1 h prior to protein
incorporation. Hydrolyzed TMOS sol (0.100 mL) was mixed with an
equal volume of pure PtNHase (1.8 mg) in 50 mM Tris–HCl, pH 7.5,
3
00 mM NaCl. The mixture was placed on ice until gelation occurred
to create PtNHase:sol–gel monoliths at the bottom of a glass vial.
Following gelation, the monoliths were washed three times with
0
4
.400 mL of 50 mM Tris–HCl, pH 7.5 (sol–gel buffer) and stored at
C overnight in 0.400 mL of the same buffer. The following day, the
◦
aged monoliths were crushed to produce a heterogeneous material
and then washed three times with 0.400 mL of the sol–gel buffer.
The buffer used to age the PtNHase:sol–gels and buffer from the
wash were tested for protein leaching by the Coomassie (Bradford)
Protein Assay Kit (Pierce).
Fig. 1. SDS-PAGE of purified PtNHase. Lane 1 purified PtNHase and lane 2 molecular
weight standards.
above. The activity of the control samples was taken as 100%.
The PtNHase:sol–gels were thoroughly washed three times with
2.6. Kinetic characterization of the PtNHase:sol–gel
0
.400 mL of sol–gel buffer to remove trypsin or ␣-chymotrypsin
The activity of the PtNHase:sol–gel biomaterial was determined
and digestion products. Then the PtNHase:sol–gel hydrogels were
assayed for catalytic using the standard assay conditions.
by measuring the conversion of acrylonitrile to acrylamide using
high performance liquid chromatography (HPLC). A 5 mL solution
◦
of 600 mM acrylonitrile in 50 mM Tris–HCl pH 7.5 at 35 C was
2.9. Activity of soluble and encapsulated PtNHase in organic
reacted with PtNHase:sol–gel biomaterial with constant shaking at
50 rpm. Aliquots of the reaction mixture (0.300 mL) were taken at
co-solvents
2
fixed-time intervals and analyzed by HPLC using a Shimadzu Shim-
Pack XR-ODS C18 reverse-phase column (3.0 mm i.d. × 75 mm)
with a mixture of 97% water: 3% methanol as the mobile phase
at 220 nm. The specific activity (U/mg) of the PtNHase:sol–gel bio-
material was calculated from the reaction rate (mmol/L/min), the
amount of the PtNHase encapsulated, and the volume of the reac-
tion. The concentration of acrylamide produced was determined
using standard curves of peak area versus known acrylamide con-
centrations.
The activity of the soluble PtNHase with 50, 70, and 100% (v/v)
methanol as the organic co-solvent toward acrylonitrile was mea-
◦
sured spectrophotometrically at 25 C as described above. Each
measurement was repeated at least three times. The hydration
of acrylonitrile using the PtNHase:sol–gel hydrogels with organic
co-solvents at 50%, 70% and 100% (v/v) was carried out in 5 mL
◦
reactions of 600 mM acrylonitrile at 25 C with constant shaking
(
250 rpm). Aliquots, 0.300 mL, of the reaction mixture were taken
at fixed-time intervals and analyzed by HPLC as described above.
2.7. Recycling experiments
2.10. Thermostability of the soluble and immobilized PtNHase
A 5 mL solution of 600 mM acrylonitrile in 50 mM Tris–HCl pH
.5 at 35 C was reacted with PtNHase:sol–gel biomaterial for 1 h
◦
The thermostability of soluble PtNHase and PtNHase:sol–gel
7
◦
after which an aliquot (0.300 mL) was taken for HPLC analysis.
The product mixture was then decanted and the hydrogel washed
with 1.2 mL of sol–gel buffer. The PtNHase:sol–gel hydrogel was
re-suspended in 5 mL of 600 mM acrylonitrile in 50 mM Tris–HCl
biomaterial was determined by incubation at 50, 60, 70, and 80 C
for 0, 30, 60, 180, 300, 420, and 600 min. After the incubation period
was completed, the residual enzyme activity of the samples was
determined using the assay conditions described above.
◦
pH 7.5 at 35 C and the reaction repeated as described above.
This process of reacting, recovering, washing, and re-suspending
the PtNHase:sol–gel hydrogel was repeated 12 additional times
resulting in 12 separate cycles with the same sample of the
PtNHase:sol–gel biomaterial.
3. Results and discussion
3.1. Encapsulation of PtNHase
Engineering of a hexa-histidine tag allowed for the isolation of
∼10 mg per liter of culture of pure PtNHase (Fig. 1) using a single
purification step. Purified PtNHase was then used for encapsulation
in tetramethylorthosilicate (TMOS) gels using the sol–gel method.
Analysis of both the buffer in which the monoliths were stored and
the buffer from all the washing steps using the Bradford assay indi-
cated that on average <0.5% of the PtNHase protein was present in
both the wash buffer and the storage buffer. These data indicate that
∼99.5% of the protein is encapsulated within the sol–gel matrix.
Under solution conditions, PtNHase catalyzes the hydration of
2
.8. Proteolytic digestion of soluble and PtNHase:sol–gels
Trypsin digestion of soluble (0.5 mg) PtNHase and
PtNHase:sol–gel material (1.8 mg of PtNHase) was performed at a
ratio of 1:1 (trypsin:PtNHase) in 50 mM Tris–HCl and 1 mM CaCl ,
pH 7.6 (trypsin reaction buffer) at 35 C for 18 h with constant agi-
tation. The ␣-chymotrypsin digestion of soluble PtNHase (0.5 mg)
and PtNHase:sol–gel material (1.8 mg of PtNHase) was performed
in 100 mM Tris–HCl and 10 mM CaCl , pH 8.0 (␣-chymotrypsin
buffer), also at a 1:1 ratio (␣-chymotrypsin:PtNHase) at 35 C for
1
2
◦
2
◦
◦
−1
acrylonitrile at pH 7.5 and 25 C with a kcat value of 1460 ± 90 s
8 h with constant agitation. Along with the digestion samples,
and a Km value of 1.3 ± 0.3 mM. Interestingly, non-proteolytically
controls that consisted of PtNHase in trypsin and ␣-chymotrypsin
reaction buffers were also incubated at 35 C for 18 h with con-
digested PtNHase:sol–gel biomaterial react readily with acryloni-
◦
◦
trile (Fig. 2) at 35 C in 50 mM Tris–HCl pH 7.5 with >90% conversion
stant agitation. Following digestion, the degraded samples of
soluble PtNHase and controls were centrifuged, cooled on ice, and
assayed for PtNHase activity using the assay conditions described
of 600 mM acrylonitrile to acrylamide in 60 min. These data indi-
cate that the kinetics of substrate turnover for sol–gel encapsulated
NHases appear to be governed by mass transport of the substrate

2
2
S. Martinez et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
6
5
4
3
2
1
00
00
00
00
00
00
0
1
00
7
5
2
5
0
5
0
Cmtp*
Cmtp+
Trypsin*
Trypsin+
0
10
20
30
40
50
60
70
80
Fig. 3. Residual activity of soluble PtNHase (*) and PtNHase:sol–gels (+) proteolyti-
cally digested with ␣-chymotrypsin (Cmtp) and trypsin. The soluble PtNHase (*) and
PtNHase:sol–gels (+) were incubated at a 1:1 ratio of enzyme:protease for 18 h at
5 C. Residual activity was measured with 100 mM (soluble PtNHase) and 600 mM
PtNHase:sol–gels) acrylonitrile in 50 mM Tris–HCl pH 7.5 at 35 C.
Time (Minutes)
◦
3
(
Fig. 2. Time course for the hydration of 600 mM acrylonitrile to acrylamide in 50 mM
Tris–HCl pH 7.5 at 35 C using PtNHase:sol–gels containing 1.8 mg of PtNHase.
◦
◦
through the porous gel to the enzyme active site and product
release. The reaction mixture was analyzed by high-performance-
liquid chromatography (HPLC) and no acrylic acid was detected
indicating that only the desired product, acrylamide, was produced.
Quantitating the reaction of acrylonitrile with PtNHase:sol–gel bio-
materials is difficult as the exact quantity of encapsulated enzyme
is not known, however, assuming that ∼99.5% of the enzyme used
was encapsulated at least 12% of the solution state activity is
A.
1
1
20
00
80
◦
retained at 35 C. Protein denaturation or inaccessibility to enzyme
buried within the sol–gel material likely results in reduced activ-
ity upon encapsulation of the enzyme. Even so, these biomolecular
nanocomposites display the expected enzymatic properties includ-
ing substrate recognition as NHases in solution.
6
4
2
0
0
0
0
50 °C
6
7
8
0 °C
0 °C
0 °C
3.2. Proteolytic digestion of soluble PtNHase and PtNHase:sol–gel
biomaterial
0
100
200
300
400
500
600
To ensure that substrate has access to fully encapsulated enzyme
and not simply protein adhered to the surface, the PtNHase:sol–gel
biomaterial was treated with trypsin or ␣-chymotrypsin to pro-
teolytically digest all surface accessible protein. Interestingly, the
PtNHase:sol–gel biomaterial retained >90% of its residual activ-
ity after digestion with either trypsin or ␣-chymotrypsin (Fig. 3)
while soluble PtNHase retained <15% of its residual activity after
digestion, indicating that surface bound enzyme is hydrolyzed, but
interior PtNHase is not. These data also indicate that substrate has
access to the PtNHase enzyme trapped in the interior of the sol–gel
biomaterial, which is an active catalyst but protected from proteo-
lytic digestion.
Time (Minutes)
B.
1
00
80
6
4
0
0
5
6
7
8
0 °C
0 °C
0 °C
0 °C
3.3. Thermostability of soluble PtNHase and PtNHase:sol–gel
biomaterial
The thermostability of soluble PtNHase enzyme and the
20
0
PtNHase:sol–gel biomaterial was evaluated over
ture range of 50–80 C. Both soluble PtNHase enzyme and
a tempera-
◦
◦
PtNHase:sol–gel biomaterial were highly stable at 50 C over
◦
0
100
200
300
400
500
600
6
00 min incubation (Fig. 4). At 60 C, the stability of the soluble
PtNHase enzyme decreases significantly after 30 min of incuba-
tion (Fig. 4A); however, the PtNHase:sol–gel biomaterial showed
greater stability at this temperature (Fig. 4B). The PtNHase:sol–gel
Time (Minutes)
Fig. 4. Thermostability of soluble PtNHase (A) and PtNHase:sol–gels (B). The residual
activity of the soluble and encapsulated after the high temperature incubation was
determined by measuring the hydration of acrylonitrile to acrylamide at 35 C.
biomaterial also showed improved stability after incubation at
◦
◦
7
0 C compared to the soluble enzyme (Fig. 4B). Interestingly,

S. Martinez et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
23
6
5
4
3
2
1
00
00
00
00
00
00
0
6
5
4
00
00
00
300
200
100
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
10 11 12 13
Reaction #
Weeks
Fig. 5. Production of acrylamide using recycled PtNHase:sol–gels. Each reaction
consisted of 600 mM acrylonitrile in 50 mM Tris–HCl pH 7.5 at 35 C, 1 h.
Fig. 6. Long-term stability of the PtNHase:sol–gels. The PtNHase:sol–gel hydrogels
◦
were used for the hydration of 600 mM acrylonitrile in 50 mM Tris–HCl pH 7.5 at
◦
3
5 C, 1 h.
neither the soluble enzyme nor the biomaterials were stable when
◦
incubated at 80 C. Based on published studies that have looked at
1
00
75
the thermostability of soluble vs. encapsulated enzymes [17,31,32],
we hypothesized that the observed improvement in thermostabi-
lity of the enzyme upon the encapsulation in the sol–gel matrix is
due to the enzyme being in a conformation that prevents unfolding
and thus its denaturation at high temperatures.
3
.4. Reusability and long-term stability of the PtNHase:sol–gel
5
2
0
5
0
biomaterial
For commercial applications, a biocatalyst must be reusable and
have long-term stability [33]. With this in mind we sought to inves-
tigate whether the PtNHase:sol–gel biomaterial could be recycled
in subsequent reactions with acrylonitrile. After every use, the
PtNHase:sol–gel biomaterial was thoroughly washed with buffer
to remove residual acrylonitrile and acrylamide then submitted to
the same reaction conditions (Fig. 5). The conversion of acrylonitrile
to acrylamide decreases linearly with ∼50% of the initial activity
remaining after thirteen cycles. Remarkably, the PtNHase:sol–gel
Sol 50/50
Encp 50/50
Sol 70/30
Encp 70/30
Fig. 7. Residual activity of the soluble (Sol) and encapsulated (Encp) PtNHase for
the conversion of acrylonitrile to acrylamide in a solution of 50/50 and 70/30
MeOH/water.
◦
biomaterial can also be stored at 4 C in buffer and used weeks later
without significant loss of activity (Fig. 6). Thus, encapsulation of
PtNHase provides enhanced stability providing a biomaterial that
can be recycled multiple times, stored, and reused.
in 70:30 MeOH:water solvent mixtures (Fig. 7). Therefore, sol–gel
encapsulation clearly affords significant stabilization against
increasing protic solvent concentrations, compared to the enzyme
solutions.
3.5. Stability of the soluble PtNHase and PtNHase:sol–gel
biomaterial in organic co-solvent
4. Conclusions
Typically, the solubility of nitriles at high concentrations
becomes problematic requiring organic solvents; however,
enzymes often denature in organic solvents. Remarkably, the
PtNHase:sol–gel biomaterial placed in methanol is able to
hydrolyze acrylonitrile to acrylamide. Since only one mole of
water is consumed in each catalytic cycle, enough water may
exist in the sol–gel or in the methanol to allow the encapsu-
lated enzyme to remain catalytic. Therefore, we examined the
hydration of acrylonitrile in 90:10 (data not shown), 70:30
and 50:50 MeOH:water solvent mixtures (Fig. 7). Greater than
In summary, in this work we present the first encapsulation of
a nitrile hydratase in a sol–gel matrix. The resulting biomaterial
(PtNHase:sol–gel) exhibits catalytic activity toward acrylonitrile
providing only the acrylamide as the product. Although the activ-
ity of the encapsulated PtNHase is diminished compared to the
soluble PtNHase, its thermal stability is markedly improved. The
hydrogels were active and stable when exposed to proteases
and the enzyme could withstand protic solvent mixtures. Addi-
tionally, PtNHase:sol–gel biomaterials were successfully used and
recycled for the hydration of 600 mM acrylonitrile to acrylamide
9
5
0% conversion of acrylonitrile to acrylamide was achieved in
0:50 MeOH:water solvent mixtures while ∼75% conversion was
◦
and remained active for more than 8 weeks at 4 C. The beauty of
achieved in 70:30 MeOH:water solvent mixtures. For comparison
purposes, soluble PtNHase exhibits only 65% of its native activity
in 50:50 MeOH:water solvent mixtures which decreases to 15%
NHase biocatalytic materials as organic synthetic tools resides in
the fact that the NHase:sol–gel materials can be cast into any shape
desired and if cast as pellets, can be added in a catalytic amount to a

2
4
S. Martinez et al. / Journal of Molecular Catalysis B: Enzymatic 100 (2014) 19–24
reaction mixture and simply filtered or decanted off after a pre-
scribed reaction time. Moreover, these materials will potentially
provide functional biomaterials capable of hydrolyzing nitriles in
a chemo, regio, and stereoselective manner from a wide variety of
nitrile substrates thus, providing synthetic chemists new avenues
to design synthetic methodologies using nitriles as starting mate-
rials.
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This work was supported by the National Science Foundation
CHE-1058357, RCH) and the Petroleum Research Fund (ACS PRF
0033-ND4, RCH). The authors gratefully acknowledge Dr. Miguel
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