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

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Enzyme immobilization, Micro-reactor, Enzymatic membrane, Anodic aluminum oxide, Lipase, Tyrosinase, L-DOPA
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.
Alternative Title
Fabrication of TA-FeⅢ/AAO Membrane Carrier for Enzyme Immobilization and Development of Micro-PFR
Alternative Author(s)
Jo Eun Yeong
일반대학원 화학공학과
Awarded Date
Table Of Contents
List of Tables ⅴ
List of Figures ⅵ
Abbreviations ⅹⅰ

제1장 서론 1
제1절 연구 배경 1
제2절 이론적 배경 4
1. 효소 (Enzyme) 4
가. Lipase 4
가. Tyrosinase 5
2. 효소 고정화 (Enzyme immobilizaition) 7
3. Anodic aluminum oxide (AAO) membrane 11
4. Tannic acid (TA) 13
5. Plug flow reactor (PFR) 15

제2장 실험재료 및 방법 17
제1절 실험재료 및 기기 17
제2절 효소고정화 담체 제조 및 특성 분석 18
1. TA-FeⅢ/AAO membrane 담체 제조 18
가. TA-FeⅢ/AAO membrane 제조 18
나. TA-FeⅢ/AAO membrane 에 효소 고정화 18
2.효소고정화 특성 19
가. 효소 고정화 19
나. 효소 정량 19
다. 효소 활성 측정 방법 19
3. 고정화 효소의 pH와 온도에 따른 활성측정 20
4. 고정화 효소의 저장안정성 20
5. 고정화 효소의 재사용성 평가 20
제3절 고정화 담체를 이용한 micro-PFR 21
1. LIP/TA-FeⅢ/AAO membrane을 이용한 micro-PFR 설계 21
2. 체류시간에 따른 전환율과 생산성 측정 21
3. Micro-PFR에서 장기간 안정성 21
제4절 TA-FeⅢ/AAO membrane에 tyrosinase 고정화 및 적용 22
1. Tyrosinase activity 측정 22
2. TA-FeⅢ/AAO membrane에 tyrosinase 고정화 22
3. 고정화 tyrosinase의 저장 안정성과 재사용성측정 23
4. TYR/TA-FeⅢ/AAO membrane을 micro-PFR 적용 23
5. Micro-PFR의 작동 안정성 및 L-DOPA 연속 생산 23

제3장 결과 및 고찰 29
제1절 TA-FeⅢ/AAO membrane 29
1. TA와 FeCl3 합성비율에 따른 효소고정화 29
2. Scanning electron microscopy (SEM) 측정 32
3. Fourier-transform infrared spectroscopy (FT-IR) 측정 35
제2절 TA-FeⅢ/AAO membrane 효소고정화 38
1. 고정화 효소의 pH와 온도에 따른 활성도 변화 38
2. 고정화 효소의 저장 안정성과 재사용성 41
제3절 TA-FeⅢ/AAO membrane기반 micro-PFR 44
1. TA-FeⅢ/AAO membrane을 이용한 micro-PFR 설계 44
2. 최적 체류시간 선정 47
3. Reactor수에 따른 전환율의 영향 49
4. Micro-PFR의 작동 안정성 51
제4절 TA-FeⅢ/AAO membrane에 tyrosinase 고정화 및 적용 53
1. EA, EAC, EAPC 법에 따른 tyrosinase 활성 53
2. Free tyrosinase와 고정화 tyrosinase의 L-DOPA 생산 56
3. 고정화 tyrosinase의 저장 안정성과 재사용성 59
4. TYR/TA-FeⅢ/AAO membrane 기반 Micro-PFR 62
가. 최적 체류시간 선정 62
나. TYR/TA-FeⅢ/AAO membrane 기반 Micro-PFR 작동 안정성 64

제4장 결론 67

References 69
조선대학교 대학원
조은영. (2020). 효소 고정화를 위한 TA-FeⅢ/AAO Membrane 담체 개발 및 Micro-PFR 설계.
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