Figure 1.
The effect of absorbing time on the enzyme loading amount (a) and effect of enzyme loading amount on the activity recovery (b).
Figure 2.
The effect of modification of support on enzyme binding.
An amount of 0.1 g MCM-41-RgPAL and MCM-41-NH-GA-RgPAL were dissolved in 10 mL Tris-HCl buffer (25 mM, pH 8.6) and stirred in a conical flask at room temperature. The residual enzyme was subsequently determined every 2 h.
Figure 3.
The FT-IR spectra of MCM-41 and MCM-41-NH2.
The FTIR spectra were recorded using NICOLET NEXUS FTIR-470 (Thermo Electron Corporation) in the range of 4000–500 cm−1.
Figure 4.
The characteristics of free Rg PAL and MCM-41-NH-GA-Rg PAL.
(a) The optimal pH of free RgPAL and MCM-41-NH-GA-RgPAL. The effects of pH were determined at 50°C using a series of buffers with various pH values. (b) The pH stability of free and immobilized RgPAL. The activity was assessed following the enzyme treatment in buffer at different pH values (5.0–12) for 12 h. The relative activity at pH 8.5 was defined as 100%. (c) The optimal temperature of free and MCM-41-NH-GA-RgPAL. (d) The thermostability of free RgPAL and MCM-41-NH-GA-RgPAL. The free RgPAL and MCM-41-NH-GA-RgPAL were incubated at 60°C for 10–60 min. The relative activity before incubation was defined as 100%. The values presented correspond to the mean values of at least three replicates.
Figure 5.
The reusability of MCM-41-NH-GA-Rg PAL.
The initial activity of MCM-NH-GA-RgPAL was measured and then compared with the activity of the used enzyme obtained after its repeated use per day. After each cycle, MCM-41-NH-GA-RgPAL was immediately filtered, washed with buffer solution and stored at 4°C.
Figure 6.
Comparison of resolution of DL-phenylalanine in a RPBR and in a batch stirred-tank reactor.
Two equal amounts of MCM-41-NH-GA-RgPAL (100 U) were loaded in a RPBR and a batch stirred-tank reactor, respectively. The substrate of 1 liter (100 mM) were fed to the RPBR with a flow rate of 8 mL min−1, or directly added in the batch stirred-tank reactor. The reaction products were periodically collected at the reactor outlet and analyzed for the conversion ratio.
Figure 7.
Effects of residence time and substrate concentration on the resolution of DL-phenylalanine.
To compare the conversion ratio and volumetric conversion rate under different operational conditions, the reactor was run for 12 h, and then the sample was obtained from the reactor analysis. (a) The effect of residence time on the conversion ratio and the volumetric conversion rate. The substrate (40 mM, 1 L) was fed into the RPBR with different flow rates to provide different residence times. (b) The effect of substrate concentration on the conversion ratio and volumetric conversion rate. The substrate (1 L) was fed into a RPBR with a flow rate 12 mL min−1.
Figure 8.
The effect of controlling pH on the resolution.
(a) Effects of pH regulation on the conversion ratio. (b) Effects of pH regulation on eeD. The substrate (100 mL, 1 L) was added to the reactor with a flow rate of 12 mL min−1. When the resolution was performed for 16 h, the pH was adjusted from 8.5 to approximately 5 to precipitate the trans-cinnamic acid from the reaction solution. The trans-cinnamic acid was removed by filtration, and then the pH of the reaction was readjusted to 8.5, and the resolution was continued.
Figure 9.
Operation stability of the RPBR.
For each batch, 100 mM of substrate was recycled through the RPBR with a flow rate 12 mL/min. At 16 h, the pH was readjusted from 8.5 to approximately 5 to precipitate the trans-cinnamic acid. After the trans-cinnamic acid was removed by filtration, the pH of the reaction was readjusted to 8.5 to continue, and each batch run for 24 h. The reaction solution was sampled for analysis every 4 h.
Figure 10.
Resolution curves of DL-phenylalanine in the small RPBR and scaled-up RPBR.
The volume of the reaction solution and the quantity of enzyme used were increased proportionally with the reactor size. The operation process in the scaled-up reactor maintained a similar residence time (0.625 h) and load of substrate (25 L of 100 mM), and the pH was regulated from 8.5 to approximately 5 to precipitate the trans-cinnamic acid at 16 h. After the trans-cinnamic acid was removed by filtration, the pH of the reaction was readjusted to 8.5, and the reaction was continued.