3
8
S. Yu et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 37–42
Currently, the hydrolysis of 3-HPN should offer an effec-
8.0) or immobilized cells containing equal amounts of wet cells
in 10 mL Tris-HCl buffer (100 mmol/L, pH 8.0) was incubated with
the substrate at 30 C with 200 rpm shaking for 10 min. A simple
(500 L) of the reaction mixture was taken and quenched with
200 L HCl (1.0 mol/L) and 300 L distilled water. The concen-
tration of 3-HP was measured by HPLC analysis. One unit of the
enzyme activity was defined as the amount of whole cells produc-
ing 1 mol of 3-HP per minute.
tive approach for the synthesis of 3-HP. However, the chemical
hydrolysis of 3-HPN requires strongly basic conditions and high
temperature, and produces hypersaline waste water. Biocatalysis
offers a “greener” alternative with mild reaction conditions and
excellent selectivity. Klempier et al. reported that 100 mmol/L of
◦
3
2
-HPN could be hydrolyzed to 3-HP by immobilized nitrilase in
4 h and the yield was 63% [26]. Bramucci et al. demonstrated that
Comamonas testosteroni 5-MGAM-4D and Comamonas testosteroni
2-1 cells could catalyze the hydrolysis of 3-HPN to 3-HP with
2
2.4. Analytical methods
a maximum substrate concentration of 1.0 mol/L after 15 h [27].
The low substrate concentrations have limited their application
on the industrial scale. This study is aimed at developing a com-
mercially feasible process for the production of 3-HP, which has
been achieved by using free or immobilized recombinant E. coli cells
harboring a nitrilase gene, with high activity for the hydrolysis of
Aliquots (100 L) were taken at different intervals, and the reac-
tions were terminated by adding 200 L HCl (1.0 mol/L) and 700 L
distilled water. The yields were determined by HPLC analysis per-
formed on an Agilent 1200 series HPLC system with an Eclipse
XDB-C18 column and monitored at wavelength of 210 nm with a
3
2
2
-HPN at high substrate concentration.
UV detector. The eluent was a mixture of phosphoric acid solution
◦
(0.05%) and methanol (95/5, v/v), column temperature was 30 C
. Materials and methods
and flow rate was 0.8 mL/min. Under these conditions, retention
time of 3-HP was 2.6 min (Fig. S3 in the Supporting information).
Aliquots (100 L) were drawn and quenched by adding 100 L
HCl (6 mol/L) and 800 L ethyl acetate. The mixture was centrifuged
at 12,000g for 1 min, and the organic phase was used to measure the
residual substrate and 3-hydroxypropionamide by GC analysis after
dried over anhydrous sodium sulfate. GC analysis was performed
using a CP-ChiraSil-DEX CB column (25 m × 0.25 mm × 0.25 m)
with helium as the carrier gas and flame ionization detector. The
.1. Materials
The recombinant strains used in current study were main-
tained in our laboratory. 3-Hydroxypropionamide was prepared as
described in the literature (for 1H and C NMR see Figs. S1 and 2
in the Supporting information) and used as standard sample for GC
analysis [28]. 3-Hydroxypropionitrile was purchased from Sigma-
Aldrich, 3-hydroxypropionic acid was purchased from TCI (Tokyo
Chemical Industry Co., Ltd.,). Sodium alginate was purchased from
Sinopharm Chemical Reagent Co., Ltd. The other chemicals were
purchased from commercial sources. The High Performance Liquid
Chromatography (HPLC) analysis was performed with an Agilent
13
◦
injector and detector temperature was set at 220 C, and the oven
◦
◦
temperature was controlled as follows: 50 C (3 min) −20 C/min
◦
−180 C (4 min). The retention times for standard samples of 3-HPN
and 3-hydroxypropionamide were 7.12 and 9.41 min, respectively.
No 3-hydroxypropionamide was detected in the reaction mixture.
1
200 series HPLC system. The GC analysis was performed on an
1
13
Agilent 7890A gas chromatography (GC) system. H and C NMR
spectra were recorded on a Bruker AVANCE-III 400 MHz NMR spec-
trometer.
2.5. Immobilization of whole cells
Freshly harvested cells (4%, w/v) were suspended in a NaCl solu-
tion (0.9%), and sodium alginate (3%, w/v) was dissolved in a NaCl
solution (0.9%). Once the alginate solution was cooled to room tem-
perature, the suspension cells were added into the solution and
thoroughly mixed. The mixture was then added dropwise from a
peristaltic pump to a stirred solution of calcium chloride (2%, w/v)
which was precooled in the ice water. After stirring for 4 h, the
formed beads were filtered and washed three times with distilled
water. Part of those was stored in fresh calcium chloride solution
2
.2. Selection of the nitrilase
The nitrilases were expressed in E. coli BL21(DE3) cells and
the recombinant strains were cultured in the Luria-Bertani (LB)
medium (containing adequate antibiotic) at the optimized condi-
tions (Table S1). Cells were harvested by centrifugation, washed
once with sodium chloride (0.9%) and cryopreserved at −20 C.
◦
◦
The biotransformation was performed by using 100 g/L
(2%, w/v) at 4 C and the other was used for chemical cross-linking.
(
wet cells weight) resting cells in 1.0 mL reaction mixtures,
The alginate beads were stirred in distilled water containing 2%
(v/v) of PEI (50%) for 20 min at the room temperature. After filtered
and washed three times with distilled water, the beads were stirred
in 2% (v/v) of glutaraldehyde solution (25%) and filtered immedi-
ately after 30 s. The chemically cross-linked alginate beads were
washed three times with distilled water and stored in fresh calcium
which contained 1.0 mol/L 3-HPN in potassium phosphate buffer
(
100 mmol/L, pH 8.0). The reaction mixtures were incubated at
◦
3
0 C and 200 rpm for 6 h.
2
.3. Preparation of whole cell biocatalysts and enzyme activity
◦
assay
chloride solution (2%, w/v) at 4 C for use in the biotransformation
reaction [29].
The plasmid pET-32a-Nit190 was transformed into E. coli BL21
DE3) cells and the recombinant strains were grown in 4 mL
LB medium containing 100 g/mL ampicillin at 37 C with shak-
ing 200 rpm for 6–8 h. The culture was inoculated into 800 mL
LB medium with 100 g/mL ampicillin, which was then incu-
bated at the same conditions. When the optical density at 600 nm
(
2.6. Optimization of biotransformation conditions
◦
For the optimization of reaction conditions, 1.0 mL (for free
cells) or 10 mL (for immobilized cells) of the reaction mixture was
used with variations as follows. The concentration of free cells
or immobilized cells which contained equal amount of wet cells
was 20 g/L, and the substrate concentration was 1.0 mol/L for opti-
mization of pH, temperature, and thermal stability. Four kinds of
buffers were used to determine the optimal pH, including citrate
buffer (100 mmol/L, pH 5.0–6.0), phosphate buffer (100 mmol/L, pH
6.0–8.0), Tris-HCl buffer (100 mmol/L, pH 7.0–9.0), and glycine-
(
OD600) was 0.6–0.8, the gene expression was induced by adding of
0
.1 mmol/L isopropyl -d-1-thiogalactopyranoside (IPTG) for 12 h
◦
at 25 C. The cells were harvested by centrifugation, washed once
◦
with 0.9% NaCl solution and stored at −20 C for use.
The activity of the free whole cells or immobilized cells was
assayed by using 3-HPN (1 mol/L) as substrate. A suspension
◦
(
20 mg/mL wcw) of cells in 1 mL Tris-HCl buffer (100 mmol/L, pH
NaOH buffer (100 mmol/L, pH 9.0–10.0), at 30 C for 1 h. The