C. Han et al. / Journal of Molecular Catalysis B: Enzymatic 115 (2015) 113–118
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2.7. Reducing the formation of 3-aminopropanamide by removal
of ammonia
The reaction was carried out at various substrate concentra-
tion ranging from 0.6 to 3.0 M which was adjusted to pH 7.3 by
fumaric acid. The wet cells of BjNIT3397 (100 mg) and aspartate
ammonia-lyase (150 mg) were added into the substrate solution
(1 mL). The bioconversion was implemented at 25 ◦C, 200 rpm for
12 h. The reaction was terminated by addition of 10% sodium car-
bonate. The conversion of 3-aminopropionitrile was measured by
HPLC analysis as described in Section 2.4.
2.8. Time course for the biotransformation and product
characterization
The biotransformation of 3-aminopropionitrile was performed
by following the same procedure in Section 2.4. The reaction vol-
ume was 20 mL with final concentration of substrate being 3 M
30 ◦C, pH 7.3 and 200 rpm for 8 h. At every half hour interval in
the first 4 h, and then every hour interval, a sample was with-
drawn from the reaction mixture, and analyzed by HPLC analysis as
described above. A quarter of the reaction mixture was derivatized
by following a literature method [28]. Di-tert-butyldicarbonate
(9.0 g, 41 mmol) and NaOH (5.0 g, 125 mmol) was added to the
stirred reaction solution (50 mL). The mixture was stirred overnight
at room temperature. The N-Boc 3-aminopropanamide (3a) was
extracted by dichloromethane (4 × 100 mL). The combined organic
extracts was dried over anhydrous sodium sulfate, the solvent was
evaporated in vacuo and the residue was purified by silica gel col-
umn chromatography to afford N-Boc 3-aminopropanamide (2a,
0.42 g, 15%) as a white solid. The water layer was acidified by satu-
rated citric acid solution to pH 3.0 and extracted with ethyl acetate
(4 × 100 mL). The combined organic extract was dried over anhy-
drous sodium sulfate, the solvent was evaporated in vacuo and the
residue was purified by silica gel column chromatography to afford
N-Boc -alanine (1a, 1.89 g, 67%) as a white solid. N-Boc -alanine
(1a) 1H NMR (400 MHz, CD3OD): ı 3.31 (t, J = 6.8 Hz, 2 H, –CH2NH–),
2.49 (t, J = 6.8 Hz, 2 H, –CH2CO2H), 1.45 ppm (s, 9H, –Boc). 13C NMR
(100 MHz, CD3OD): ı = 174.07, 156.89, 78.76, 35.98, 33.89, 27.38.
N-Boc 3-aminopropanamide (2a) 1H NMR (400 MHz, CD3OD): ı
3.28–3.36 (m, 2H, –CH2NH–), 2.40 (t, J = 6.8 Hz, 2H, –CH2CONH2),
1.45 ppm (s, 9 H, -Boc). 13C NMR (100 MHz, CD3OD): ı 175.33,
156.92, 78.73, 36.49, 35.23, 27.35.
The biotransformation of 3-aminopropionitrile by nitrilase
and aspartate ammonia-lyase followed the same procedure as
described above. The pH of a solution of 3-aminopropionitrile (4.2 g,
final concentration of 3 M) was adjusted to 7.3 by adding fumaric
acid (4.0 g, final concentration 1.8 M) and the final volume was
20 mL. The wet cells of BjNIT3397 (2.0 g, 1000 U) and aspartate
ammonia-lyase (3.0 g, 3297 U) were added into the reaction mix-
ture and the reaction was implemented at 30 ◦C, 200 rpm for 8 h.
The samples were withdrawn from the reaction mixture at different
time intervals, and analyzed by HPLC analysis as described above.
A quarter of the reaction mixture was purified by cation exchange
resin and a mixture of ammonium salts of -alanine and l-aspartic
acid (1 + 3, 1.91 g). 1H NMR (400 MHz, D2O): ı 3.69 (dd, J = 8.7,
3.6 Hz, 1H, l-aspartic acid, –CH–), 3.01 (t, J = 6.7 Hz, 12 H, -alanine,
–CH2NH2), 2.60 - 2.68 (m, 1H, l-aspartic acid, –CH2–), 2.47 (dd,
J = 17.1, 8.7 Hz, 1H, l-aspartic acid, –CH2–), 2.40 ppm (t, J = 6.7 Hz,
12H, -alanine, –CH2CO2H). 13C NMR (100 MHz, D2O): ı 178.54
(-alanine, –CO2H), 177.98 (l-aspartic acid, –CH2CO2H), 175.65
(l-aspartic acid, –CHCO2H), 52.44 (l-aspartic acid, –CH–), 37.70
(l-aspartic acid, –CH2–), 36.57 (-alanine, –CH2NH2), 33.94 (-
alanine, –CH2CO2H). The product mixture (1 + 3) was derivatized
with 1-fluoro-2-4-dinitrophenyl-5-l-alanine amide and analyzed
Fig. 1. pH dependence of the enzyme activity of nitrilase BjNIT3397 towards 3-
aminopropionitrile (600 mM substrate, 25 ◦C for 2 h).
by LC–MS. The mobile phase was a mixture of water (0.5% formic
acid) and methanol in the volume ratio of 64:36 and the flow
rate was 1.0 mL/min (see Supplementary Information). The high-
resolution MS (ESI) of FDAA derivatized l-Aspartic acid (3b): m/z:
calcd for C13H16N5O9+: 386.0948 [M + H]+, found: 386.0942. The
high-resolution MS (ESI) of FDAA derivatized -alanine (1b): m/z:
calcd for C12H16N5O7+: 342.1050 [M + H]+, found: 342.1049.
3. Results and discussion
3.1. Nitrilases selection
lases available in our laboratory were carried out at the substrate
concentration of 5 mM and TLC analysis showed that a few nitri-
lases catalyzed the hydrolysis of 3-aminopropionitrile to -alanine
(Table 1). However, when the substrate concentration increased to
50 mM, only BjNIT3397 remained the activity. As such, BjNIT3397
was selected for the further studies.
3.2. Effect of reaction pH and temperature
The nitrilase activity of the whole cell biocatalyst toward 3-
aminopropionitrile at different pH were studied by measuring the
conversion of the substrate. Concentrated hydrochloric acid (12 M)
was used to adjust the pH of the aqueous substrate solution to the
desired value. It had been found that the enzyme was active in this
pH range and the optimal pH was around 7.0 (Fig. 1). This is slightly
different from the pH profile of the purified BjNIT3397 toward
range with optimum from pH 7.0 to 8.0 [26].
The nitrilase activity of the whole cell biocatalyst toward
3-aminopropionitrile was measured at different temperatures
ranging from 20 to 70 ◦C. The optimal reaction temperature was
about 40 ◦C (Fig. 2), slightly lower than that (45 ◦C) of the purified
enzyme toward phenylacetonitrile [26].
3.3. The formation of 3-aminopropanamide
The nitrilase BjNIT3397 catalyzed the hydrolysis of 3-
aminopropionitrile at the substrate concentration up to 3.0 M
under the optimal reaction conditions. However, as the sub-
strate concentration increased,
a significant amount of 3-
aminopropanamide (about one third of the products at 3 M
substrate concentration) was observed in the product mixture,
demolishing the production of -alanine (Fig. 3). The by-product