Fig.S1
2.5. Preparation of immobilized
enzyme
D-amino acid dehydrogenase
immobilized in ZIF-8 (DAADH/ZIF-8) were prepared by mixing zinc nitrate
solution (0.16 M, 500 μL), enzyme solution (500 μL) and
2-methylimidazole (0.16 M, 500 μL) together and then shaking at 4 ℃, 200
rpm, 30 min. The mixing solution was centrifugated at 4 ℃, 8000 rpm, 15
min. The supernatant was collected to measure the protein concentration
for further determination of enzyme loading rate. The precipitate was
washed with deionized water three
times, and then resuspended it with PBS buffer (200 mM, pH 7.4).
D-amino acid dehydrogenase immobilized in hybrid materials of ZIF-8 and
RGO (DAADH/ZIF-8/RGO) was prepared by mixing zinc nitrate solution (0.16
M, 500 μL), enzyme solution (500 μL), 2-methylimidazole (0.16 M, 500 μL)
and RGO (1 mg/mL,500 μL). After shaking at 4 ℃, 200 rpm for 4 h, the
solution was centrifuged at 4 ℃, 8000 rpm, 15 min. The supernatant was
collected and measure the protein concentration for further
determination of enzyme loading rate. The precipitate was washed with
deionized water three times, and then resuspended it with PBS buffer
(200 mM, pH 7.4).
One-step separation and immobilization of enzyme from crude solution was
investigated. D-amino acid dehydrogenase immobilized in hybrid materials
of ZIF-8, RGO and Ni (DAADH-ZIF-8/RGO/Ni-DAADH) was prepared by mixing
zinc nitrate solution (0.16 M, 500 μL), NiCl2 (0.08 M,
500 μL), enzyme solution (500 μL), 2-methylimidazole (0.16 M, 500 μL)
and RGO (1 mg/mL,500 μL), and followed by the shaking, centrifugation
and resuspending as above.
2.6. Morphology
characterization
Morphology analysis of immobilized enzyme was carried out by scanning
electron microscopy with a Zeiss Sigma scanning electronic microscopy
(SEM) (Carl-Zeiss AG, Germany). JEOL JFC 1600 (JEOL, Tokyo, Japan) was
used to spray platinum on the sample to make it conductive.
2.7. Reusability
Reusability of
immobilized enzyme was studied by
recycling the enzymatic reaction for seven times. At the ending of each
reaction, the immobilized enzyme was re-collected by centrifugation at
8000 rpm for 15 min (4 °C) and washed with PBS buffer (pH 7.4). Initial
enzyme activity of first cycle was set as 100 %, and the remaining
activity was counted for percent form (%) compared with that of the
first reaction.
2.8. Thermostability
The enzyme was incubated in water bath at 50 °C for 10 h. The enzyme
activity was measured by taking samples every 2 h (at 0,2,4,6,8,10 h).
The relative enzyme activity was calculated by setting the enzyme
activity of the unincubated enzyme solution as 100%.