J. Li et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 24–28
25
Due to the advantages of nanofibrous membranes, here we
2.4. Effect of temperature and pH on aminoacylase activity
investigated the properties and activities of aminoacylases on PVA
nanofibrous memberanes. After optimizing the immobilization
procedure we moved forward to test and optimize aminoacylase
separation d- and l-theanine. This procedure will have much util-
ity in the industrial processes of not only theanine enantiomers but
other chiral amino acids.
The effect of temperature on the activities of free and immo-
bilized aminoacylase was examined for the range of 29–72 C at
◦
pH 7. The effect of pH was tested over the range pH 5–9 at
◦
52 C.
2.5. Stability of immobilized aminoacylase
2
. Materials and methods
The thermostability of free and immobilized aminoacylase
was determined by measuring the residual enzyme activity, as
described above, every other day during storage in phosphate
buffer (0.1 M, pH 7) at 52 C.
2
.1. Materials
◦
PVA (with a degree of polymerization of 1750 ± 50) was pur-
The free and immobilized aminoacylase were stored in phos-
chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China). Aminoacylase (ACY, EC3.5.1.14) and d/l-theanine were
purchased from Shanghai Hanhong Chemical Co., Ltd. Standard
l-theanine was provided by Tokyo Kasei Kogyo Co., Ltd. Glutaralde-
hyde (GA) (50%, AR) was obtained from Shanghai Aibi Chemical Co.,
Ltd. Genipin (GP) (MW = 226.23, 98% by high-pressure liquid chro-
matography) was purchased from Linchuan Zhixin Bio-Technology
Co., Ltd. Polyethylene glycol diglycidyl ether (PGDE) was supplied
by Yifu Chemical Materials Co., Ltd.
◦
◦
phate buffer (0.1 M, pH 7) at 25 C and 4 C for 6 d and 30 d,
respectively. The residual enzyme activities were measured at dif-
ferent time intervals.
The reusability of immobilized aminoacylase was studied by
measuring the residual enzyme activities repeatedly for 5 times.
After each reaction, the enzyme was washed with fresh phosphate
buffer (0.1 M, pH 7) and the retention activity was performed under
optimal conditions.
3. Results and discussion
2.2. Preparation of PVA/aminoacylase nanofibrous membranes by
electrospinning
3.1. Characterization of nanofibrous membranes
◦
PVA (0.7 g) was dissolved in 8.33 mL deionized water at 95 C
In order to characterize the PVA electrospun nanofibrous
over a period of 2 h, followed by stirring with 0.3 g aminoacylase at
membranes with and without aminoacylase (ACY), SEM images
were taken (Fig. 1). The nanofibrous membranes have a uniform
morphology with an average diameter of ∼200 nm (Fig. 1a). The
average diameter of the nanofibers increased to ∼300 nm when
aminoacylase (ACY) was added (Fig. 1b). This is probably because
the PVA/ACY mixture becomes viscous after the formation of
intermolecular hydrogen bonds between PVA hydroxyl groups
and ACY amino groups. From Fig. 1c–e, it is clear that crosslinkers
caused the formation of nonuniform nanofibers with different
diameters.
◦
2
5 C to form a homogeneous solution. Then, a crosslinker (GA, GP,
or PGDE) was dropped into the solution at a final concentration of
.5%, 0.5%, or 2% (v/v), respectively. For electrospinning, the mixture
0
was transferred into a 20-mL syringe equipped with a metal nee-
dle that was connected to a high-voltage power supply (GDW-A;
Beijing Institute of High Voltage Electrical and Mechanical Tech-
nology, Inc., China). A syringe pump was used to control the flow
rate at 0.8 mL/h, and electrospinning was performed at 15 kV at a
distance of 12 cm between the needle tip and the collector. The
◦
membranes were collected for 4 h and then dried overnight at 4 C
FTIR spectra for nanofibrous membranes of pure PVA, PVA/ACY,
and PVA/ACY/crosslinkers were acquired to characterize the immo-
bilization of aminoacylase on the membranes (Fig. 2). Pure PVA
under vacuum.
The morphologies of the PVA/aminoacylase nanofibrous mem-
branes were characterized with scanning electron microscopy
−
1
exhibited a wide band from 3100 to 3600 cm that was assigned
to O–H stretching and intermolecular hydrogen bonds [28]. All
(
SEM, XL-30; Philips, Eindhoven, Netherlands) and Fourier trans-
form infrared spectroscopy (Nicolet 5700, Thermo Fisher Scientific,
USA).
−
1
immobilized ACY membranes exhibited a new peak at 1652 cm
indicating O C–NH bonds formed between the enzyme and PVA
20]. Thus, ACY was immobilized on the PVA membranes via chem-
ical bonding.
,
[
2.3. Activity assay of free and immobilized aminoacylase
Aminoacylase activity was measured by determining the pro-
3.2. Effect of temperature and pH on aminoacylase activity
duction of l-theanine from N-acetyl-d/l-theanine via the ninhydrin
colorimetric method described by Rosen [27], with slight modi-
fications. To resolve theanine enantiomers, a reaction mixture of
Fig. 3 shows the aminoacylase activity over the temperature
◦
◦
range of 29–72 C and pH 5–9. Under optimal conditions (42 C
and pH 7) enzyme activity retention was maintained at 100%. With
immobilization, the ACY activity retention decreased to 49.2–70.5%
N-acetyl-d/l-theanine (0.02 M, pH 7) and aminoacylase solution
◦
(
5 mg/mL) was incubated at 52 C for 30 min. The chromogenic
reaction was initiated by addition of 0.2 mL acetate buffer (2 M,
pH 5.4) and 0.2 mL ninhydrin solution (1%, w/v) to a 0.2 mL resolu-
tion mixture. This mixture was heated in boiling water for 15 min,
followed by cooling to room temperature. Afterwards, 0.2 mL was
mixed with 3 mL 60% ethanol for the colorimetric measurement at
◦
relative to that of free ACY at 42 C, most likely due to activity
loss during the immobilization process. When the temperature was
◦
increased to 52 C, the free ACY activity decreased to 90% of that at
◦
4
2 C. In contrast, all immobilized ACYs had their highest activi-
◦
◦
ties at 52 C. Thus, the optimal temperature shifted from 42 C for
free ACY to 52 C for immobilized ACY, suggesting that immobilized
5
70 nm. A standard calibration curve was used to determine the
◦
aminoacylase activity. One activity unit (U) of aminoacylase was
defined as the amount of enzyme required to generate 1 mol of
l-theanine per minute. The retention of aminoacylase activity was
defined as the ratio of the activity of the immobilized enzyme to
that of the same amount of free enzyme. All activity assays were
performed in triplicate.
enzymes could remain in a stable conformation and remain robust
at high temperatures.
For free ACY, pH 7 was optimal, whereas pH 8 was opti-
mal for the immobilized ACYs, with 52.9–77.8% activity retention.
Thus, the immobilized enzyme required a higher pH for opti-
mal activity than that of the free enzyme. This is most likely