효소 고정화를 위한 TA-FeⅢ/AAO Membrane 담체 개발 및 Micro-PFR 설계

Abstract

Enzymatic membrane micro-PFR was developed using anodic aluminum oxide (AAO) membrane coated with tannic acid (TA) and iron ions (FeⅢ) complex. TA has large number of hydroxyl (-OH) groups, which can be functionalized for enzyme attachment to achieve a higher enzyme to carrier ratio. AAO membrane has high surface area for the retention of enzyme, therefore it offers an attractive platform for enzyme immobilization. Lipase (from Aspergillus niger) has been used as a model enzyme to be immobilized on the TA-FeⅢ/AAO membrane. Fourier-transform infrared (FT-IR) spectroscopy was used to characterize the chemical features of prepared TA-FeⅢ /AAO membranes. Scanning electron microscopy (SEM) was used to observe the surface morphologies (i.e., pore size, thickness). When TA and FeCl3 concentration ratio was 2:1, the enzyme immobilization yield of TA-FeⅢ/AAO membrane was the highest (72.04%). The relative activity of the immobilized lipase with TA-FeⅢ coating is much more stable than that without coating, and it is even higher than that of free lipase without reuse. After 30 days of storage and reuse 9 times, more than 80% of the activity was retained with TA-FeⅢ coating, while the relative activity was 60% without coating. The plug flow reactor was developed using TA-FeⅢ/AAO membrane (V=700 uL). The lipase was immobilized and used for bioconversion. With the change of flow rate (from 1 to 5 mL/min), the largest productivity was reached 13.57 mol/h·L at 4 mL/min. After 80 days of continuous operation, the enzyme activity was remained over 90%. Based on the success of lipase immobilization, immobilization of tyrosinase on TA-FeⅢ/AAO membrane using EAPC method was performed. After 30 days of storage time, more than 70% of the activity was remained using TYR/TA-FeⅢ/AAO membrane, while that of free tyrosinase was 36%. TYR/TA-FeⅢ/AAO membrane retained 65% of its initial activity after 6 times reuse. The production of L-DOPA was monitored while applying the TYR/TA-FeⅢ/AAO membrane micro-PFR. The highest productivity of L-DOPA was reached 0.0862 mol/h·L at 0.1 mL/min. After 30 days of continuous operation, immobilized tyrosinase showed 70% of residual activity at 20OC. As a result, when micro-PFR was applied using LIP/TA-FeⅢ/AAO and TYR/TA-FeⅢ/AAO membrane, the rate of reaction was faster about 134 times and 15 times rather than batch reactor, respectively. This research exhibits the potential application of micro-PFR in the bio-catalytic field.