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니켈 foam을 이용한 우수한 고정화 재료 합성 및 고정화효소 특성 분석

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Author(s)
사방지
Issued Date
2020
Abstract
생명 공학, 재료, 화학 및 다른 표면개질 방법의 지속적인 개발로 인해 새로운 재료들이 계속 등장하고 있어 고정화 기술 연구를 위한 담체 원료들이 많이 개발되고 있다. 니켈 폼 (Ni-foam)은 가격이 저렴하고 다루기 쉬우며 스펀지와 같은 다공성 구조로 인해 베이스 담체 재료로 매우 적합하다. 따라서 본 연구에서는 Ni-foam을 담체 재료로 사용하였고 polyaniline nanofibers (PANI), polyaniline magnetic particles (PAMP), tannin iron ion complex (TA@FeCl3_6H2O), polydopamine (PDA)을 포함한 다양한 폴리머 재료로 Ni-foam 표면을 코팅하였다. 이들은 작용기를 가지고 있어 화학적 가교법으로 효소를 고정화하기가 더 쉽다. PANI, PAMP, TA@FeCl3_6H2O, 및 PDA 합성 단계에서 Ni-foam을 넣어 Ni-foam 표면을 코팅하였다. 이전에, 이러한 폴리머 물질들은 효소 고정화 재료로서 분산 된 입자 형태로 사용된다. 이러한 방법은 회수가 어렵다는 단점이 있다. 그러나 Ni-foam을 사용할 경우 반응 용액으로부터 쉽게 회수가 가능하고, 반응을 쉽게 제어할 수 있다는 장점이 있다. 고정화 된 효소 및 담체 특성은 fourier transform infrared (FTIR-ATR) spectrophotometer, scanning electron microscope (SEM), 및 UV/Visible spectrophotometer를 사용하여 특성화되었다.
목적으로 하는 폴리머들이 Ni-foam 표면에 합성이 되었는지 FTIR-ATR 분석법을 통해 확인하였고, SEM 이미지를 통해 표면이 개질된 Ni-foam에 효소가 고정화되었음을 확인할 수 있었다.
고정화 된 lipase (LIP) 및 glucose oxidase (GOx)의 안정성을 분석 하였다. 그 결과 순수한 Ni-foam에 고정화된 GOx의 활성은 한 달 후에 거의 사라졌다. 그러나 PANI, PAMP, TA@FeCl3_6H2O 및 PDA로 코팅된 Ni-foam에 GOx를 고정화 하였을 때 잔류 활성은 각각 50.9%, 70.4%, 72.1%, 73.4%였다. 순수한 Ni-foam에 고정화 된 LIP의 초기 활성은 PAMP, PANI, PDA로 코팅된 것보다 높았지만 안정성은 좋지 않았다. TA@FeCl3_6H2O상의 고정화 된 LIP는 가장 안정적이고 우수한 잔류 효소 활성을 가졌으며, 이어서 PDA, PAMP였다. 1 개월 후, 순수한 Ni-foam 과 PANI, PAMP, TA@FeCl3_6H2O 및 PDA로 코팅된 Ni-foam에 고정화된 효소의 잔류 활성은 각각 83.1%, 73.3%, 93.4%, 90.1% 및 98.3%였다.
600 μL 용량의 PFR 마이크로 반응기가 개발되었고, 관형 반응기 안에 Ni-foam에 고정화된 효소가 사용되었다. 실험 결과, PDA-GOx 및 PAMP-GOx는 양호한 작동 안정성을 보였다. PDA-GOx를 사용한 포도당 전환율은 172 시간 후에 84.6±4.7%에서 18.4±2.3%로 감소한 반면, PAMP-GOx는 82.1±1%에서 22.2±4.4%로 감소했다. LIP의 경우, 기질 4-NPB의 가수 분해 생성물이 pH에 의해 크게 영향을 받아 pH를 6.5로 고정하고 마이크로 PFR을 사용하여 실온에서의 작동 안정성을 보았다. 그 결과 24 일 연속 작동 후에도 전환율이 변하지 않았다.
요약하면, Ni-foam의 표면 개질은 효소 반응 공정의 안정성을 증가시킬 뿐만 아니라 공정을 경제적으로 가능하게 하였다.|With the continuous development of biotechnology, materials, chemistry and other modification methods, new materials continue to emerge, enriching the carrier sources of immobilization technology researches. Nickel foam (Ni-foam) was used as the carrier material, because of its being inexpensive, easy to handle and sponge-like porous structure makes it well suited as a base carrier material. In this study, the surface of Ni-foam was coated with various polymer materials, including polyaniline nanofibers (PANI), polyaniline magnetic particles (PAMP), and tannin iron ion complex (TA@FeCl3_6H2O), polydopamine (PDA). They have functional groups, thereby making it easier to immobilize the enzyme with the chemical cross-linking method. PANI, PAMP, TA@FeCl3_6H2O, and PDA were synthesized in one step and subsequently coated on Ni-foam. Previously, these polymer materials were used in the form of dispersed particles as enzyme immobilizers. However, after Ni-foam was coated with these materials, it was easier to separate them from the reaction solution and control the reaction. The immobilized enzymes and its carrier properties were characterized by using fourier transform infrared (FTIR-ATR) spectrophotometer, scanning electron microscope (SEM), and UV/Visible spectrophotometer.
The FTIR-ATR spectroscopy results showed that the polymer particles synthesized by one-step method were our desired polymer materials and provided a direct proof for the synthetic process of these polymer materials. The SEM images showed the various nanofibrous forms which had increased surface area for enzyme immobilization.
The stabilities of immobilized lipase (LIP) and glucose oxidase (GOx) were analyzed . The results showed that, the activity of GOx immobilized Ni-foam almost disappeared after a month. However, the residual activities of PANI, PAMP, TA@FeCl3_6H2O, and PDA were 50.9%, 70.4%, 72.1%, 73.4%, respectively. The initial activity of immobilized LIP on bare Ni-foam was higher than that of PAMP, PANI, PDA coatings, but the stability was not good. Immobilized LIPs on TA@FeCl3_6H2O was the most stable and had the best residual enzyme activity, followed by PDA, PAMP. After one month, the residual activities of immobilized enzymes on bare Ni-foam, PANI, PAMP, TA@FeCl3_6H2O, and PDA were 83.1%, 73.3%, 93.4%, 90.1%, and 98.3%, respectively.
A PFR micro reactor with a capacity of 600 μL was developed, and the nickel foam with immobilized enzyme was used in a tubular reactor. The experimental results showed that PDA-GOx and PAMP-GOx have good operational stabilities. The glucose conversion with PDA-GOx decreased from 84.6±4.7% to 18.4±2.3% after 172 hours, while that of PAMP-GOx from 82.1±1% to 22.2±4.4%. For LIP, the hydrolysate of substrate 4-NPB was greatly affected by pH, so the pH was fixed to 6.5, and micro PFR was used to detect its operation stability at room temperature. The results showed that the conversion did not change after 24 days continuous operation of.
In summary, the modification of Ni-foam did not only increase the stability of the enzyme reaction process, but also make the process economical feasible.
Alternative Title
Synthesis of Novel Immobilized Materials Based on Nickel Foam and Study on Properties of Immobilized Enzymes
Alternative Author(s)
XIE FANGZHI
Department
일반대학원 화학공학과
Advisor
이중헌
Awarded Date
2020-02
Table Of Contents
Contents
List of Tables IV
List of Figures V
ABSTRACT VIII
Ⅰ. Introduction 1
A. Immobilization technology 1
B. Immobilization carrier materials 5
1. Traditional immobilization carriers 5
2. New immobilization carriers 5
C. Carrier materials-PANI, PAMP, TA@FeCl3_6H2O, PDA 7
D. Research outline 13
Ⅱ. Materials and Methods 14
A. Materials 14
1. PANI and PAMP 14
2. TA@FeCl3_6H2O 14
3. PDA 14
4. Enzyme immobilization and activity assay 14
B. Methods 15
1. Preparation for PANI, PAMP 15
2. Preparation for TA@FeCl3_6H2O 15
3. Preparation for PDA 15
4. Immobilization of enzymes on different carriers 16
5. Characterizations 19
6. Enzyme loading test 19
7. Enzyme activity assay 21
8. Storage stability of immobilized enzyme 26
9. Application of Micro-PFR with immobilized enzymes 26
a) Micro-PFR process using immobilized GOx 28
(1) Production of gluconic acid using Micro-PFR process 28
(2) Effect of pH, temperature and substrate concentration on Micro-PFR
process 32
(3) Response surface analysis of Micro-PFR 35
(4) Study on the operational stability of immobilized enzymes with
Micro-PFR 39
b) Micro-PFR process using immobilized LIP 40
Ⅲ. Results and Discussion 42
A. Morphology and size of immobilized GOx/LIP 42
B. Surface chemistry analysis 45
C. Enzyme loading analysis 50
D. Storage stability analysis 52
E. Stability analysis of Micro-PFR 54
1. Operational stability of immobilized GOx 54
2. Operational stability of immobilized LIP 56

Ⅳ. Conclusion 58
References 60
초록 67
Degree
Master
Publisher
조선대학교 화학공학과
Citation
사방지. (2020). 니켈 foam을 이용한 우수한 고정화 재료 합성 및 고정화효소 특성 분석.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/14102
http://chosun.dcollection.net/common/orgView/200000279086
Appears in Collections:
General Graduate School > 3. Theses(Master)
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