Figure 1.
The reactions catalyzed by GA3-oxidase.
In each metabolic reaction, the modification is highlighted in red.
Figure 2.
Characterizations of the META-silica hybrid monolithic column.
(A) – (C) SEM images. (A) ×1,000 wide-view, (B) ×4,000 close-up-view, and (C) ×15,000 close-up-view. (D) The N2 isothermal plot with the inset showing the pore-size distribution. (E) The effect of flow rate on the back pressure of the monolithic column. (F) Van Deemter plot of the height equivalent to a theoretical plate as a function of flow rate. Experimental conditions: column, poly(META-co-DVB-co-EDMA) monolithic column (30-cm long, 100 µm i.d., 360 µm o.d.); UV detection wavelength, 254 nm for acrylamide, 214 nm for thiourea and benzene; mobile phase used in (E), ACN; mobile phase used in (F), ACN/H2O (60/40, v/v).
Figure 3.
Investigation of the separation mechanisms for five GAs.
(A) The effects of FA content on the retention factor (k) of GAs. (B) The effects of ACN content on the retention factor (k) of GAs. Experimental conditions: column, poly(META-co-DVB-co-EDMA) monolithic column (30-cm long, 100 µm i.d., 360 µm o.d.).
Figure 4.
Extracted ion chromatogram of five GA standards.
Shown in inset is the total ion chromatogram of five GA standards. Experimental conditions: column, poly(META-co-DVB-co-EDMA) monolithic column (30-cm long, 100 µm i.d., 360 µm o.d.); flow rate, 800 nL/min; mobile phase, ACN/H2O/FA (60/40/0.6, v/v/v). Order of peaks: 1. GA1, 2. GA20, 3. GA53, 4. GA4, 5. GA9.
Figure 5.
Optimization of the mass spectrometry detection conditions of GA1.
(A) Full-scan spectrum of GA1. (B) Full-scan spectrum of GA1 with optimized ESI source conditions. Experimental conditions: 1 µg/mL GA1 were infused in mobile phase (ACN/H2O/FA, 60/40/0.6, v/v/v) at a flow rate of 3 µL/min.
Table 1.
Linearity, LODs and LOQs of GAs obtained by cLC-MS method.
Figure 6.
The kinetics study of GA3-oxidase.
(A) Michaelis–Menten plots of recombinant GA3-oxidase in E. coli. cell lysate. (B) Lineweaver–Burk plots for GA9. (C) Lineweaver–Burk plots for GA20. Experimental conditions: GA9 or GA20 was incubated for 15 min at 30°C with E. coli. cell lysate and cofactors.
Figure 7.
Extracted ion chromatogram of the catalytic products and substrates of GA3-oxidase using 5 mg of rice embryos.
Experimental conditions: column, poly(META-co-DVB-co-EDMA) monolithic column (30-cm long, 100 µm i.d., 360 µm o.d.); flow rate, 800 nL/min; mobile phase, ACN/H2O/FA (60/40/0.6, v/v/v). Order of peaks: 1. [2H2]GA20, 2. [2H2]GA53 (I.S.), 3. [2H2]GA4, 4. [2H2]GA9.