2. Materials and methods
2.1 Structures of DAADH
DAADH originating fromUreibacillus thermosphaericus was selected (PDB ID: 5gz6). An
expression vector for DAADH_5gz6 with polypeptide linker (amino acid
sequence: (RTHRK)4) was constructed by amplifying the
DAADH_5gz6 gene fragment infusion by PCR. The amplified gene fragment
was digested with Nde I and Xho I and ligated with the expression vectorE.coli BL21(DE3)/pET28a to generate pET28a/ DAADH_5gz6pep, which
was then transformed into Escherichia coli strain Rosetta (DE3)
(Novagen). The experiment used the plasmid extraction and gel recovery
kit produced by Takara. Added 500 µL of recombinant expression strains
to 500 µL 50% sterile glycerin, mixed and stored at -20 ℃.
Cells were cultivated overnight
at 37 ℃ and 200 r/min in 10 mL of LB medium containing 50 μg/L
kanamycin. The culture solution was transferred to 100 ml of LB medium
containing 50 μg /L kanamycin by volume fraction of 1 %, and cultured
at 37 ℃ for 2-3 h until the optical density at 600 nm reached 0.6-0.8.
Then added isopropyl-β-d-thiogalactopyranoside (IPTG) with final
concentration as 1 mM, and continued to cultivate for 12 h at 25 ℃.
2.2. Chemicals
Kanamycin and isopropyl-beta-D-thiogalactopyranoside (IPTG) were
purchased from Trans-Gen Biotech (Shanghai, China). NADPH, ionic
liquids, phenylpyruvate and all other chemicals were purchased from
Sigma Chemical Company (Tianjin, China).
RGO is prepared from our laboratory. Methods: Add 0.05 g ascorbic acid
to 1 mL, 1 mg/mL GO dispersion and stirred at room temperature for 12 h.
The solution was centrifuged at 8000 rpm for 15 min and then was
resuspended with deionized water, then centrifuged and resuspended in
same condition, finally obtained 1 mg/mL RGO dispersion.
2.3. Enzyme extraction and purification
Cells were harvested by centrifugation, then suspended in potassium
phosphate buffer (pH 7.4,10 mM), and disrupted by untrasonication (200
W, 3 s, 85 times) in ice water bath. The cell debris was removed by
centrifugation (8000 rpm) for 20 min.
The DAADH obtained by
recombination-induced expression was labeled with His-tag. Ni-NAT His
Bind Resin column (Shanghai Qixing Company) was then equilibrated with
10 column volumes of loading buffer (20 mM Tris, 0.25 M NaCl, pH 7.4).
Crude enzyme solution was collected by filtration through the membrane
filter (0.45 μm) and loaded into binding column. The column was then
washed with ten column volumes of binding buffer (20 mM imidazole, 20 mM
Tris, 0.25 M NaCl, pH 7.4). Finally, target protein was eluted with
elution buffer (200 mM imidazole, 20 mM Tris, 0.25 M NaCl, pH 7.4) and
was desalted and concentrated by Millipore ultrafiltration tube, the
content of pure enzyme protein was 0.169 mg/mL.
2.4. Enzyme assay
The activities of DAADH and
immobilized DAADH were determined by using phenylpyruvate as substrate
in NH4Cl-NH3·H2O buffer
(200 mM, pH 9.5). The activity was measured by detecting the NADPH
concentration at 340 nm by Infinite 200 PRO spectramicroplate reader
(TECAN). The initial velocities was calculated as consumption rate of
NADPH when the conversion rate of substrate was below 5%. Specific
activity was recorded as U/mg enzyme. The amount of enzyme consumed for
generation or consumption of 1 μmol of NADPH per minute was defined as
one unit of enzyme activity. Specific activity (U/mg) is defined as the
ratio of enzyme activity (U/mL) to protein concentration (mg/mL) at 30
℃. Parallel experiments were repeated four times. The relative activity
was expressed as the maximal value of enzyme activity at a certain pH or
temperature as 100%.