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재조합 glutamate decarboxyalse를 이용한 -Aminiobutyric acid (GABA) 생산공정 최적화

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Author(s)
김의진
Issued Date
2014
Abstract
Recently, bio-based fuels and polymers from renewable biomass have drawn much attention due to the increasing environmental concerns and concern over the limited nature of fossil resources. For example, polylactic acid(PLA) made from corn has been considered as a good alternative to petroleum-based plastic and has widely been employed for general purpose plastics because of several desirable properties such as biodegradability, biocompatibility, compostability, and low toxicity to humans. However, most biopolymers cannot be applied in high-value-added areas such as automobile manufacture and electronic devices because the mechanical and thermal properties of biopolymers are not able to meet high performance requirements of such applications.
Nylon 4 is four-carbon polyamide suitable for application as an engineering plastic due to its superior thermal and mechanical properties. Contrary to other nylon polymers, nylon 4 is heat-resistant, biodegradable, biocompatible and compostable. Nylon 4 is synthesized by ring-opening polymerization of petroleum-derived 2-pyrrolidone. Because 2-pyrrolidone can be prepared from gamma-aminobutyric acid(GABA), GABA produced from renewable resources such as L-glutamic acid, which is eventually used as the monomer for the synthesis of nylon 4, could broaden the industrial applications of GABA.
In this study, we have demonstrated the hybrid process composed of biological and chemical processes, in which biomass-derived 2-pyrrolidone was used for the synthesis of nylon 4. High concentration of GABA was synthesized from MSG with recombinant E. coli expressing glutamate decarboxylase and then GABA was converted into 2-pyrrolidone that was finally used for the synthesis of nylon 4.

The fermentation process was developed to improve the GAD production using recombinant E. coli(BL21). The effects of ammonium sulfate, magnesium sulfate, glucose concentration, three different feeding methods and pH on the production GAD were investigated. Results showed that the production of GAD was the highest and reached 830.5 units when glucose 40 g/L, (NH4)2SO4 6 g/L, KH2PO4, 3 g/L, K2HPO4 3 g/L, yeast extract 1 g/L and 350 rpm, 7 vvm, and pH 5.5.

pH controlled batch reactor and bubble column reactors have been developed in this research. They were used to produce high concentration of GABA and to determine optimal pH for GABA production. Glutamate decarboxylase (GAD) was isolated from recombinant E. coli and used for GABA production from monosodium glutamate (MSG). pH control was inevitable because the pH increased with MSG consumption. GAD showed highest activity at acidic conditions at pH 5.5 but the optimal pH for GABA production was pH 6.0. When 1.5 mole of MSG was used as reactant, the 1.05 mole of GABA was produced after 10 hrs batch reaction. Using bubble column reactors, 80 % of MSG was converted to GABA for 6 hrs reaction and 1.2 mole of GABA was produced.

2-Pyrrolidone was synthesis with high pressure reactor using produced GABA. The sodium chloride in the GABA solution was removed using acetone precipitation to improve 2-pyrrolidone synthesis. The bulk polymerization process using 2-pyrrolidone has been developed for the production of Nylon 4. The NMR results confirmed that the produced polymer was Nylon 4.
Alternative Title
Optimization of GABA production process using recombinant glutamate decarboxylase
Alternative Author(s)
Eui Jin Kim
Department
일반대학원 화학공학과
Advisor
이중헌
Awarded Date
2015-02
Table Of Contents
목차
List of Figures V
List of Table IX
ABSTRACT XI
제 Ⅰ 장 Glutamate decarboxylase의 생산공정 개발 1
Ⅰ- 1 장 서론 1
1. 이론적 배경 1
1.1. Gamma-Aminiobutyric acid(GABA) 1
1.2. Glutamate decarboxylase(GAD) 3
1.3. 회분식 배양 7
1.4. 미생물 성장 및 대사산물 생산속도론 11
1.5. 비생장속도(Specific growth rate)와 비활성(Specific activity) 13
Ⅰ- 2 장 실험 재료 및 방법 14
1. 시약 및 기기 14
2. 배양조건 14
2.1. 균주 및 보존 14
2.2. 배지조성 14
3. 실험방법 16
3.1. 배양방법 16
3.2. Glutamate decarboxylase(GAD) 분리 16
4. 분석방법 17
4.1. 건조 균체량과 질소원, 탄소원 농도측정 17
4.2. GAD activity 측정 19
4.3. Glutamate 와 GABA 농도측정 20
4.4. 배양 조건에 따른 반응 표면 분석 22
Ⅰ- 3 장 실험결과 23
1. 배양 조건 23
1.1. Ammonium sulfate 농도의 영향 23
1.2. Magnesium sulfate 농도의 영향 28
1.3. Monosodium glutamate(MSG)농도의 영향 32
1.4. Glucose농도의 영향 35
1.5. pH의 영향 39
1.6. 배양조건에 따른 반응표면 분석 43
Ⅰ- 4 장 결론 45
제 Ⅱ 장 GABA 생산공정 개발 46
Ⅱ - 1 서론 46
1. 이론적 배경 46
1.1. 생체고분자(Biopolymer) 46
1.2. 효소반응 속도론 47
1.3. 효소 반응기 50
2. 국내외 연구동향 54
Ⅱ - 2 실험 재료 및 방법 56
1. 시약 및 기기 56
2. 효소 반응기 56
2.1. pH 조절이 가능한 반응기 56
2.2. 기포탑 반응기(Bubble column reactor) 58
Ⅱ - 3 실험결과 60
1. GAD 반응 특성 60
1.1. MSG 농도 변화에 따른 GABA 생산특성 60
1.2. Cofactor 농도 변화에 따른 GABA 생산 62
1.3. 효소반응 pH 변화에 따른 GABA 생산 특성 66
1.4. 결정형 GABA의 생산 69
1.5. 연속식 pH제어 반응기의 반응조건확립 72
1.6. 연속식 pH제어 반응기의 반응표면분석 82
2. GABA의 생산을 위한 반응기 scale-up 86
2.1. 분사형태의 영향 86
2.2. 반응기 교반형태의 영향 93
2.3. Bubble column reactor의 효소반응 속도론 96
Ⅱ - 4 결론 99
제 Ⅲ 장 GABA를 이용한 Nylon 4 합성공정개발 100
Ⅲ - 1 서론 100
1. 이론적 배경 100
1.1. 나일론(Nylon) 100
1.2. 나일론 4(Nylon 4) 102
1.3. 2-Pyrrolidone 103
1.4. 음이온 개환중합(Anionic ring-opening polymerization) 105
Ⅲ - 2 실험 재료 및 방법 106
1. 2-Pyrrolidone 분석 106
2. 2-Pyrrolidone 합성 107
3. Nylon 4 합성 108
Ⅲ - 3 실험결과 109
1. 2-Pyrrolidone 합성 109
2. Nylon 4 합성 118
Ⅲ - 4 결론 126
참고문헌 128
Degree
Doctor
Publisher
조선대학교
Citation
김의진. (2014). 재조합 glutamate decarboxyalse를 이용한 -Aminiobutyric acid (GABA) 생산공정 최적화.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/12363
http://chosun.dcollection.net/common/orgView/200000264796
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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