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논문 기본 정보

자료유형
학위논문
저자정보

임희선 (인천대학교, 인천대학교 일반대학원)

지도교수
서명지
발행연도
2017
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인천대학교 논문은 저작권에 의해 보호받습니다.

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이 논문의 연구 히스토리 (3)

2017

Studies on γ-aminobutyric acid production by Enterococcus faecium JK29 and Lactobacillus brevis HYE1 isolated from traditional fermented foods : 전통 발효식품으로부터 분리한 Enterococcus faecium JK29와 Lactobacillus brevis HYE1의 γ-aminobutyric acid 생산에 관한 연구
임희선 생명과학과 2017.01 학위논문
Enhanced Production of Gamma-Aminobutyric Acid by Optimizing Culture Conditions of Lactobacillus brevis HYE1 Isolated from Kimchi, a Korean Fermented Food
임희선 ,  차인태 ,  노성운 외 2명 Journal of Microbiology and Biotechnology 2017.01 학술저널

2016

Optimization of γ-Aminobutyric Acid Production by Enterococcus faecium JK29 Isolated from a Traditional Fermented Foods
임희선 ,  차인태 ,  이현진 외 1명 한국미생물·생명공학회지 2016.01 학술저널

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This study performed culture optimization for gamma-aminobutyric acid (GABA) production of Enterococcus faecium JK29 and Lactobacillus brevis HYE1 isolated from kimchi, a Korean traditional fermented food. E. faecium JK29 and L. brevis HYE1 showed GABA producing activity in screening via thin-layer chromatography (TLC) and GABA assay, resulting respectively 1.56 mM and 14.64 mM of GABA production in MRS media containing 1% (w/v) L-monosodium glutamate (MSG) at 30˚C for 48 h. Also, it was revealed that E. faecium JK29 had rare glutamate decarboxylase gene (gad) in in further screening through genetic analysis. For enhancement of GABA production by E. faecium JK29 and L. brevis HYE1, the effects of culture conditions containing carbon and nitrogen sources, initial MSG concentration and initial pH on GABA production were evaluated by optimization using one-factor-at-a-time (OFAT) strategy. As the results, the optimal conditions for GABA production by E. faecium JK29 were determined to be 0.5% (w/v) sucrose, 2% (w/v) yeast extract, 0.5% (w/v) MSG and initial pH of 7.5, resulting in 14.86 mM of GABA production. Also, when L. brevis HYE1 was cultivated in optimized MRS medium including 2% (w/v) maltose, 3% (w/v) tryptone, 1% (w/v) MSG and initial pH of 5.0, GABA production reached 18.97 mM of maximum GABA production. Furthermore, culture conditions of L. brevis HYE1 were further optimized via response surface methodology (RSM) in order to obtain more accurate optimal conditions. RSM optimization resulted 21.44 mM of predicted maximum GABA production in optimal culture conditions that modified MRS medium containing 2.14% (w/v) maltose, 4.01% (w/v) tryptone, 2.38% (w/v) MSG and initial pH of 4.74 at 30˚C for 48 h. The reproducibility experiments that conducted under these optimized conditions resulted in GABA production of 18.76 mM. Moreover, the glutamate decarboxylase (GAD) of L. brevis HYE1 was cloned and overexpressed in Escherichia coli to compare the properties of recombinant GAD with previously reported GAD, resulting in approximately 50 kDa of purified protein. As the results of the characterizations of recombinant GAD from L. brevis HYE1 (rGADLbHYE1), the optimal range of pH and temperature for the purified GAD activity were respectively 3.6-4.4 and 55-60˚C. Also, the purified GAD activity was decreased by adding AgNO3 as the inactivator of GAD activity. The purified GAD reacted only with L-monosodium glutamate as substrate and the kinetic parameter including Km value and Vmax value for purified GAD were 4.99 mM and 0.224 mM/min, respectively.
#Gamma-aminobutyric acid (GABA) #Enterococcus faecium JK29 #Lactobacillus brevis HYE1 #Optimization #Expression

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ABSTRACT ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????ⅰ
Table of Contents ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ⅲ
List of Figures ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ⅵ
List of Tables ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ⅷ
Chapter 1. INTRODUCTION ?????????????????????????????????????????????????????????????????????????????????????????????????????? 1
1.1. Gamma-aminobutyric acid ?????????????????????????????????????????????????????????????????????????????????????? 1
1.2. Lactic acid bacteria ????????????????????????????????????????????????????????????????????????????????????????????????? 2
1.3. Optimization for industrial application ??????????????????????????????????????????????????????????????????? 5
1.4. Objective of research ?????????????????????????????????????????????????????????????????????????????????????????????? 9
Chapter 2. MATERIALS and METHODS ????????????????????????????????????????????????????????????????????????????????? 10
2.1. Materials ??????????????????????????????????????????????????????????????????????????????????????????????????????????????? 10
2.1.1. Fermented food samples, bacterial strains and vectors ??????????????????????? 10
2.1.2. Culture media and conditions ?????????????????????????????????????????????????????????????? 10
2.1.3. Primers design ????????????????????????????????????????????????????????????????????????????????????? 11
2.1.4. Reagents and kits?????????????????????????????????????????????????????????????????????????????????? 11
2.2. Methods ????????????????????????????????????????????????????????????????????????????????????????????????????????????????? 13
2.2.1. Isolation and cultivation ?????????????????????????????????????????????????????????????????????? 13
2.2.2. Genetic analysis ??????????????????????????????????????????????????????????????????????????????????? 13
2.2.2.1. Sequence analysis of the 16S rRNA genes ???????????????????????? 13
2.2.2.2. Sequence analysis of the GAD gene ?????????????????????????????????? 14
2.2.3. GABA assay ???????????????????????????????????????????????????????????????????????????????????????? 14
2.2.3.1. Thin-layer chromatography analysis ?????????????????????????????????? 14
2.2.3.2. Quantitative analysis ?????????????????????????????????????????????????????????? 15
2.2.3.3. Gas Chromatography-mass spectrometry ?????????????????????????? 15
2.2.4. Culture optimization ???????????????????????????????????????????????????????????????????????????? 16
2.2.4.1. OFAT-based optimization ?????????????????????????????????????????????????? 16
2.2.4.2. RSM-based optimization ??????????????????????????????????????????????????? 17
2.2.5. Cloning of the gad gene from L. brevis HYE1 ??????????????????????????????????? 20
2.2.6. Overexpression of GAD in E. coli ?????????????????????????????????????????????????????? 21
2.2.7. Purification of GAD-(His)6 fusion protein ?????????????????????????????????????????? 21
2.2.8. SDS-PAGE analysis ???????????????????????????????????????????????????????????????????????????? 22
2.2.9. Bradford assay ????????????????????????????????????????????????????????????????????????????????????? 22
2.2.10. Characterization of GAD ??????????????????????????????????????????????????????????????????? 23
Chapter 3. RESULTS and DISCUSSION ???????????????????????????????????????????????????????????????????????????????? 24
3.1. Isolation of GABA-producing LAB ????????????????????????????????????????????????????????????????????? 24
3.2. Cell growth and GABA production ?????????????????????????????????????????????????????????????????????? 28
3.2.1. Enterococcus faecium JK29 ???????????????????????????????????????????????????????????????? 28
3.2.2. Lactobacillus brevis HYE1 ????????????????????????????????????????????????????????????????? 28
3.3. Culture condition optimization for GABA production ????????????????????????????????????????? 37
3.3.1. Enterococcus faecium JK29 ???????????????????????????????????????????????????????????????? 37
3.3.1.1. Effect of carbon sources on the GABA production ??????????? 37
3.3.1.2. Effect of nitrogen sources on the GABA production ???????? 37
3.3.1.3. Effect of MSG concentration on the GABA production ???? 40
3.3.1.4. Effect of initial pH on the GABA production ???????????????????? 42
3.3.2. Lactobacillus brevis HYE1 ????????????????????????????????????????????????????????????????? 43
3.3.2.1. Effect of carbon sources on the GABA production ??????????? 43
3.3.2.2. Effect of nitrogen sources on the GABA production ?????????43
3.3.2.3. Effect of MSG concentration on the GABA production ??? 46
3.3.2.4. Effect of initial pH on the GABA production ???????????????????? 48
3.3.2.5. Futher optimization using by RSM ???????????????????????????????????? 48
3.4. Overexpression and purification of the recombinant GAD ?????????????????????????????????? 53
3.5. Properties of the recombinant GAD ????????????????????????????????????????????????????????????????????? 53
Chapter 4. CONCLUSION ????????????????????????????????????????????????????????????????????????????????????????????????????????? 60
REFERENCES ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? 62
국문 초록 ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? 71
ACKNOWLEDGEMENT ?????????????????????????????????????????????????????????????????????????????????????????????????????????? 73

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