Escherichia coli와 Streptococcus thermophilus 균주의 글루탐산 탈탄산효소 분비 발현 및 GABA 생성에 의한 생물학적 활성

국문 초록 (Abstract)

Gamma-amino butyric acid는 인간의 뇌와 눈에 풍부하게 발현되는 4개의 탄소로 이루어진 비단백질 아미노산이며, 동물에서 고혈압과 이뇨 작용을 하는 주요 억제성 신경전달물질로 작용한다. Gamma-amino butyric acid는 식품 및 의약품에서 생리활성 성분의 잠재력으로 인해 동물신경의 이완을 촉진하고 신경 긴장을 완화하는 기능성 식품의 성분으로 활용되었다. 따라서 본 연구에서는 여러 대장균 균주들에서 (E. coli DH5α, E. coli W3110, E. coli JM109) GadA 및 GadB 유전자(gadA 및 gadB)를 T-vector에 클로닝하고, 염기서열 분석에 의해 GenBank에 등재되어 있는 서열과 비교하며, 각 균주의 gadA 및 gadB의 효과적인 발현을 위하여 T7 promoter를 함유하는 pT7-7 발현시스템에 유전자들을 도입하여 각 균주간의 GAD (GadA 및 GadB)들의 발현율 차이 및 효소 활성을 비교 분석 한다. 또한 발현 최적화 조건을 확립하여 대량생산에 의한 굴절체를 확보한 다음, 부분정제 과정을 통한 효소 순도를 향상시켜 GABA 전환율을 비교 분석하고, 가장 효율적인 GAD를 확보하였다. 마지막으로 전환 생성된 GABA의 생물학적 활성을 조사하여 산업적 응용범위 확대를 위한 기초 및 응용 자료를 도출하려 하였다. 우선적으로 GenBank에 등재 되어있는 GAD 염기서열을 [E. coli strain. K-12 substrain. MG1655 (GenBank accession No. NC000913 및 NP_417974.1)] 참고하여 특이서열의 primer set를 합성하고 주형으로는 E. coli W3110, DH5α, 및 JM109 균주들의 colony 또는 염색체 DNA를 사용하여 PCR 증폭에 의해 GAD 유전자들 (gadA 및 gadB)을 확보하고 pGem-T easy vector로 클로닝하여 DNA 염기서열 분석에 의해 도입된 gadA 및 gadB 유전자들을 최종적으로 확인하였고, T-gadA 및 T-gadB (4.4 kb)로 명명하였다. 각 균주들 간의 염기서열은 gadA의 경우에는 서로 일치하는 서열을 가지고 있으나, gadB의 경우에는 W3110 유래의 경우 GenBank gadB (Accession No. BAA15163.1)의 서열과 비교 할 때 401번째 아미노산 leucine401 (CTG)이 proline401 (CCG) 으로 바꼈고, 또한 444번째 아미노산 serine444 (TCT)이 serine444 (TCC)으로 silent mutation 된 것으로 나타났다. Glutamate decarboxylase 유전자의 CDS는 1,401 bp로 구성되며 466개의 아미노산에 대한 정보를 가지며 분자량은 51.2 kDa으로 추정되었다. 각 균주로부터 GadA 아미노산 서열과 GadB 서열은 466개 아미노산 중 5군데, 즉 GadA의 경우Gln3, Leu5, Leu6, Phe9, 및 Ala22, GadB의 경우 Lys3, Gln5, Val6, Leu9, 및 Ser22에서만 차이가 나며 나머지 부분에서는 동일한 서열을 갖는 것으로 분석되었다. Glutamate decarboxylase isoform (GadA 및 GadB)들의 균주간 발현율 및 효소 활성 차이를 알아보기 위하여 강력한 T7 (T7 bacteriophage) promoter를 지니는 pT7-7 vector (2.43 kb)로 gadA 및 gadB 유전자들을 도입하여 재조합 발현벡터를 구축하고, gadA 및 gadB 유전자가 in frame하에 도입된 것을 확인하였고, pT7-gadA 및 pT7-gadB (3.87 kb)로 명명하였다. W3110 유래 gadA 및 gadB는 pT7-gadA4와 pT7-gadB14, DH5α 유래 gadA 및 gadB는 pT7-gadA11와 pT7-gadB2로, JM109 유래 gadA 및 gadB는 pT7-gadA2와 pT7-gadB7으로 명명하였다. 대장균 W3110으로부터 유래된 gadA 및 gadB를 포함하는 발현벡터, pT7-gadA4 (형질전환체 A4-1), pT7-gadA18 (형질전환체 A18-1), pT7-gadB14 (형질전환체 B14-1)의 경우 전체 단백질 중 차지하는 GadA 및 GadB 비율은 30℃에서는 8-15% 정도로 분석되었고, 37℃에서는 6-9% 정도로 densitometer 측정에 의해 분석되었다. 대장균 DH5α로부터 유래된 gadA 및 gadB를 포함하는 발현벡터 pT7-gadA11의 형질전환체 (A11-1, A11-2), pT7-gadB2의 형질전환체 (B2-1, B2-2)의 경우 전체 단백질 중 차지하는 GadA 및 GadB 비율은 30℃에서는 16-21% 정도로 분석되었고, 37℃에서는 8-12% 정도로 densitometer 측정에 의해 분석되었다. 대장균 JM109로부터 유래된 gadA 및 gadB를 포함하는 발현벡터 pT7-gadA2의 형질전환체 (A2-1, A-2), pT7-gadA16의 형질전환체 (A16-1, A16-2), pT7-gadB7의 형질전환체 (B7-1, B7-2)의 경우 전체 단백질 중 차지하는 GadA 및 GadB 비율은 30℃에서는 19-22% 정도로 분석되었고, 37℃에서는 12-13% 정도로 densitometer 측정에 의해 분석되었다. 따라서, 발현 시 온도는 W3110, DH5α에서와 마찬가지로 30℃가 적절한 것으로 판단되며, 또한 배양시간도 24 h 보다는 6 h가 발현에 더 적합한 것으로 나타났으며, W3110와 DH5α 유래 유전자들의 발현 보다는 평균적으로 효율이 더 좋은 것으로 보이지만, 그 차이는 그렇게 크지 않으므로 배지 및 배양 조건. 발효기, 탄소 및 질소원, mineral, aeration, impella 회전속도 등 최적화에 의해 발현량을 증가 시킬 수 있을 것이다. Glutamate decarboxylase에 의한 GABA 전환율은 JM109 유래 gadA에 대한 (pT7-gadA2-1) supernatant에서 GABA전환율은 100% 이며, pellet의 경우도 100%를 나타내고, gadB에 대한 (pT7-gadB7-1) supernatant에서 GABA전환율은 100% 이며, pellet의 경우 100%를 나타내므로 JM109 유래의 효소 GadA 및 GadB의 활성은 supernatant 및 pellet에서 100% 작동하는 것으로 나타났다. 항산화 활성을 위한 GadA 효소는 JM109유래의 유전자를 포함하는 pT7-gadA2-1 벡터를 함유하는 E. coli BL21(DE3)pLysE에서 생산하였으며 (1,250 μg/mL), GadB 효소는 높은 GABA 전환 활성을 보이는 DH5α 유래의 pT7-gadB2-1 벡터를 함유하는 E. coli BL21(DE3)pLysE에서 발현 생산하였다 (1,457 μg/mL). 대조군으로 사용한 ascorbic acid의 경우 50% 정도의 항산화활성을 보였으며, 반면에 GadA의 경우는 53%, GadB의 경우에는 60% 정도의 항산화 활성을 보여 대조군보다 각각 3% 및 10% 정도의 항산화 활성이 증가한 것으로 나타났다. 세포생존율은 positive control로 사용한 GABA standard (5 mg/mL)의 경우 세포 생존율을 16% 복구시켰으며, 부분정제한 GadB (5 mg/mL)에 의해 전환된 GABA를 30 μL (150 μg) 처리하였을 때도 15% 정도 세포생존율이 복구되는 것으로 확인되었다. 따라서 GABA의 생리적 활성 중에서 비록 세포 수준이지만 제1형 당뇨에서 파괴되는 β-cell의 생존복구율을 증가 시킬 수 있는 가능성이 있음을 시사한다. 또한 NO 생성량의 억제효과는 positive control로 사용한 GABA standard 처리구에서는 STZ 처리로 당뇨를 유발하였을 때보다 산화질소의 발생량이 4배 정도 감소하였다. 또한 GABA (by GadB from E. coli BL21(DE3)pLysE/pT7-gadB2-1)를 30 μL (150 μg GABA) 처리 하였을 때 STZ 처리시 산화질소 발생량의 4.96배 정도 감소하는 것으로 나타났다. 따라서 대장균에서 발현 생산한 GadA 및 GadB의 경우 발현 최적화에 의해 대량생산을 꾀하고, 정제과정을 단순한 방법으로 개선하고 입증된 여러 생리활성들을 잘 활용한다면 산업화에 적용 가능성이 높은 잠재력을 나타낼 수 있다고 평가한다. Streptococcus thermophilus는 GAD (gadB)와 glutamate/GABA antiporter (gadC) 관련 유전자를 지니며, S. thermophilus 염색체부터 gadB유전자의 클로닝, GAD의 단백질 발현 및 분비, GABA 생성능과 생물학적 활성을 확인하기 위해 primer set 합성과 함께 PCR에 의해 유전자를 증폭하였다. 유전자 gadB의 CDS는 1,377 nucleotide로 구성되어 459 아미노산에 대하여 암호화하며 예상 분자량은 52.6 kDa이다. 배양액에 효소를 분비하여 직접적으로 glutamate를 GABA로 전환시키는 것이 효소 생산 및 전환 수율 향상을 위하여 효율적이므로 고초균에서 분비능이 확인되고, alkaline protease promoter (aprE-P, 0.45 kb), signal sequence (aprS, 87 bp) 및 리보솜결합서열 (GGAGAGGG)을 갖는 pRBAS 분비벡터에 서열 분석에 의해 확인된 gadB 유전자를 직접적으로 cloning하여 pRBAS-GadB 재조합벡터(7.83 kb)를 구축하고 B. subtilis LKS87에 형질 전환하여 재조합벡터를 함유하는 형질전환체를 확보하였다. 재조합체를 LB배지에서 시간별(12 ~ 48 h)로 배양한 다음, 발효액을 수집하여 SDS-PAGE 및 His-tag에 대한 항체를 사용하여 western blot에 의해 분비된 GAD를 확인하였으며 분비 정도는 전체 분비단백질 중 10 - 12% 정도였으며 배양시간에 따른 균체 증가에 의한 전체 분비된 단백질은 증가하였지만, 분비된 GAD가 차지하는 비율은 배양시간 및 형질전환체에 따라 비슷한 수준으로 분비되었다. 분비 효율적인 면에서 최적화 실험이 추가로 진행되어야 할 필요성은 있지만 활성적인 GAD가 분비되었다는 것을 간접적으로 확인하였다. Glutamate decarboxylase 효소에 의한 GABA 생성율을 paper chromatography에 의해 측정한 결과 GAD가 분비된 배양액의 경우 기질로 사용한 MSG를 약 16% 정도 GABA로 전환시켰으며, cell pellet을 파쇄하여 PBS buffer로 용출하여 분석하였을 때는 약 43% 정도의 GABA로 전환을 보였다. 이는 아직 분비되지 않고 세포내에 남아있는 GAD가 존재하는 것을 의미하며 신호서열 (aprS)의 교체 및 추정의 GAD 자체 분비서열 제거에 의한 분비효율이 개선될 필요성을 보이는 것이다. Glutamate decarboxylase에 의해 전환된 GABA의 생물학적 활성은, ABTS+ 라디칼을 소거하는 항산화 활성이 약 55%로, 일반적으로 알려진 ascorbic acid (50%)보다 좀 더 향상 된 것으로 나타났으며, 당뇨세포주의 제1형 당뇨모델에서 28.2% 정도의 세포 생존복구율을 보였고, 또한 STZ 처리에 의해 생성된 NO 가스 생성을 78.7% 정도 억제함으로써 항당뇨 효과의 가능성을 보였다. 그러나 아직 분비 효율의 최적화에 의한 발현 및 분비율 증가와 분비된 효소활성 증진을 위한 개선이 필요하며 또한 산업적 응용 가능성을 확장하기 위하여 가축 및 반려동물의 항스트레스 제제인 GABA를 함유하는 사료첨가제 개발도 앞으로 중점적으로 연구해야 할 과제로 남는다.

다국어 초록 (Multilingual Abstract)

Gamma-amino butyric acid (GABA) is produced from monosodium glutamate (MSG) via enzymatic conversion by glutamate decarboxylase (GAD). Gamma-amino butyric acid is a four-carbon non-protein amino acid that is abundantly expressed in the human brain and eye. In mammals, it acts as a major inhibitory neurotransmitter, inducing hypertension and diuresis. Since the low activity of cell preparations has been a major limitation to commercial enzyme synthesis, it is worthwhile to explore methods to obtain a cost-effective recombinant enzyme with high GABA synthesizing activity. Recent studies have shown that GABA is effective in promoting hypotension and anxiety relief, along with acting as a diuretic, antidiabetic, and antidepressant in humans and other mammals. Food and drug research on GABA in humans is being actively conducted worldwide, but research on livestock is limited. In this study, to compare genetic characteristics and enzyme activity of the GADs from various Escherichia coli (E. coli) strains, gadA and gadB genes were amplified using specific primer set, cloned into pGEM-T-Easy vector (T-vector), and the gene sequences were analyzed. The sequences of nucleotide and amino acid are composed of 1,401 bp CDS (coding sequence) and 466 amino acids, respectively and putative molecular weight is expected to be 51.2 kD. And then, the genes (gadA and gadB) were cloned into pT7-7 expression vector, transferred in E. coli BL21 (DE3) pLysE strain, and the expression was optimized according to culture conditions. After partial purification, GABA conversion and its physiological activity was assessed.
In experiment 1, E. coli W3110, DH5α, and JM109 strains showing highest GABA production were selected as the target strains. It was found that two isoforms of GAD, encoded by gadA and gadB, were present in E. coli. Above all, gadA and gadB genes were cloned into pGem-T-easy vector after PCR amplification from E. coli colony or chromosome as template, right clones were screened and selected, and finally the clones were named to T-gadA and T-gadB (3.87 kb) as followings: from E. coli W3110; T-gadA4, T-gadB14, T-gadA18; from DH5α: T-gadA11 and T-gadB2, T-gadB-18, P-gadB-7; from JM109 : T-gadA2, T-gadB7, T-gadA16. Among nucleotide sequences of E. coli W3110 gadA and gadB, the sequences of gadA show no difference between strains as that of GenBank (Accession No. NP_417974.1), but the sequence of gadB was changed at 2 positions of amino acid sequence from leucine401 (CTG) to proline401 (CCG) and silent mutation from serine444 (TCT) to serine444 (TCC), referred to gadB (Accession No. BAA15163.1). Otherwise, the sequences of gadA and gadB for E. coli DH5α and JM109 were identical to those of E. coli strain. K-12 substrain. MG1655 (GenBank accession No. NC000913 and NP_417974.1). By multiple alignment for amino acid sequences of GadA and GadB from E. coli W3110, DH5α, JM109 with the sequences of GenBank, the GadA sequence show no difference between strains. And also, the difference between GadA and GadB show only at the positions of 5 amino acid ; that is, Gln3, Leu5, Leu6, Phe9, and Ala22 of the GadA were changed to Lys3, Gln5, Val6, Leu9, and Ser22 of GadB and the rest of them was identical as a result of analysis. To compare different expression levels and enzyme activities of isoforms, intracellular expression of GadA and GadB in E. coli was carried out, the inclusion body was partially purified, and enzyme activity was analyzed by GABA conversion efficiency. The gadA and gadB genes (1.4 kb) cloned into T-vector were obtained after digestion of NdeI and BamHI and introduced into pT7-7 expression vector containing bacteriophage strong T7 promoter. After screening and selection of the right clones, the putative clones were sequenced, the DNA sequence in frame confirmed, and named to pT7-gadA and pT7-gadB (3.87 kb), respectively. The expression vectors such as pT7-gadA4, pT7-gadA18, pT7-gadB14 were constructed from gadA and gadB of W3110 ; pT7-gadA11, pT7-gadB2, and pT7-gadB18 from DH5α; pT7-gadA2, pT7-gadA16, and pT7-gadB7, respectively. The recombinant vectors were transferred into E. coli BL21(DE3)pLys, in which bacteriophage T7 RNA polymerase gene is cloned in chromosome of the strain (lysogen) under control of lac promoter, expressed by induction of IPTG and initiate transcription of target genes (gadA and gadB). When the expressed proteins by IPTG induction were fractionated on 12% SDS-PAGE and percentage of interest proteins for total cell proteins was measured using densitometer, in case of gadA and gadB originated from W3110, the proteins were expressed to be approximately 8-15% at 30℃ and 6-9% at 37℃, indicating that expression levels of GadA or GadB showed better condition at 30℃ and 6 h than 37℃ and 24 h. In DH5α, the levels also showed bettert 30℃ and 6 h (21-22%) than 37℃ and 24 h (8-12%). Finally, in case of gadA and gadB originated from JM 109, the proteins were expressed to be approximately 19-22% at 30℃ and 12-13% at 37℃. Accordingly, it is judged that gadA and gadB originated from JM109 showed slightly better than those of W3110 and DH5α, but expression efficiency may be improved by culture conditions (such as carbon and nitrogen sources, temperature, aeration, fermenter capacity, impella rotatary speed, mineral..etc). To detect the expressed GadA and GadB immunologically, the synthesized oligomer of His-tag (composed of 6 histidine, 5'-CACCATCACCATCACCAT-3') was fused to 3' end of the coding sequence of gadA and gadB. The GadA and GadB was expressed as 6 His additional type of existing GadA and GadB, which can be detected using anti-histidine antibody by western blot analysis. The GABA conversion rate by glutamate decarboxylase was investigated using recombinant GadA and GadB. The supernatant of cell lysates [E. coli BL21(DE3)pLysE containing pT7-gadA2-1 (gadA originated from E. coli JM109)] showed 100% GABA production from monosodium glutamate as substrate and the cell pellet suspended in PBS buffer also showed 100% conversion rate. In case of GadB originated from E. coli JM109, it is appeared to be 100% conversion rate and the pellet was same as above (100% conversion). Accordingly, GadA and GadB originated from JM109 have powerful enzyme activity in both supernatant and pellet. To investigate antioxidant activity, GadA was produced in E. coli BL21(DE3)pLysE containing pT7-gadA2-1 (gadA from JM109) (1,250 μg/mL) and GadB produced in E. coli BL21(DE3)pLysE harboring pT7-gadB2-1 (gadB from DH5α)(1,457 μg/mL). Ascorbic acid as a control showed 50% antioxidant activity, otherwise the activities of GadA and GadB was 53% and 60%, respectively. The activities were increased to 3% and 10% than the control. In insulinoma RINm5F cells, type I-like diabete was induced by streptozotocin and samples were treated to measure cell viability. The viability was restored by GABA standard (5 mg/mL) as positive control, on the other side the partial purified GadB (5 mg/mL) also increased the cell viability to 16% at concentration of 30 μL (5 mg/mL), indicating that the possibility to restore viability of β-cell destroyed in Type I diabetes is existing even though cell level at present. And to inhibit nitric oxide (NO) production, GABA was administrated in insulinoma cell, RINm5F after diabetes induction. When GABA standard was treated, NO production was decreased to 4-fold and the partial purified GABA (by GadB from E. coli BL21(DE3)pLysE/pT7-gadB2-1) also reduced ro 4.96- fold of NO production yield inside the cells. Accordingly, GadA and GadB produced in E. coli BL21(DE3)pLysE will furthermore be optimized to produce high yield of the protein and purification process will be simplified sooner or later. In my opinion, it is estimated that this study will contribute to industrialize and commercialize with high potentials.
In experiment 2, Since the low activity of cell preparations in E. coli has been a major limitation to commercial enzyme synthesis, it is worthwhile to look for methods to obtain a affordable recombinant enzyme with high GABA synthesizing activity. In order to maximize production of GABA using the culture broth without purification in Bacillus subtilis, the gene (gadB) first was obtained from chromosome of Streptococcus thermophilus (S. thermophilus) expressing the GAD with appropriate amount. The primer set with restriction enzyme sites and His-tag was designed by referring to the DNA sequence of glutamate decarboxylase enrolled in GenBank (DQ871217.2), amplified and cloned into pGEM-T-Easy vector. The right clone was screened and characterized by DNA sequencing analysis. As a result, the DNA sequence was different from that of GenBank in codons encoding 4 amino acids (Tyr189, Gly282, Ser301, and Met 425) by point mutations, which were changed to Cys189, Asp282, Gly301, and Thr425, respectively. To secret the GAD in culture medium, the recombinant vector (pRBAS-GadB, 7.83 kb) containing the gadB gene was constructed using pRBAS secretion vector with alkaline protease promoter (aprE-P, 0.45 kb) and signal sequence (aprS, 87 bp) and then transferred into Bacillus subtilis LKS87. The secretion levels of GAD were analyzed by SDS-PAGE and western-blot and the level in B. subtilis LKS87 harboring the pRBAS-GadB vector was increased to approximately 10-12% of total secreted proteins. The conversion of MSG (monosodium glutamate) to GABA was measured by paper chromatography using the culture broth and PBS-eluted cell pellet. As a result, the culture broth shows 16% GABA conversion rate from MSG, whereas the pellet shows higher conversion (~43%), indicating that the expressed GAD was not sufficiently secreting into the culture broth caused by the inefficient processing of aprS signal sequence or presence of self signal sequence of the GAD gene. By measuring the ABTS+ scavenging ability, it was confirmed that the GABA converted by GAD showed higher antioxidant activity (55%) than that (50%) of ascorbic acid used as positive control. The survival recovery rate of STZ-treated RINm5F cells was increased to 28.2% by the GABA treatment and also, production of NO was drastically reduced with 78.7% in STZ-treated RINm5F cells, suggesting that the GABA possess antidiabetic and antioxidant activities. Finally, although GABA is showing various biological activities, it might be needed to optimize secretion efficiency for industrial applications.

목차 (Table of Contents)

• Ⅰ. 초록 1
• Ⅱ. 실험 1. Escherichia coli에서 Glutamic acid decarboxylase (GadA & GadB) 발현 최적화 및 생리활성검정 6
• 1. 서론 7
• 2. 재료 및 방법 11
• 1) 재료 및 시약 11
• 2) Bacterial strains, plasmid 및 medium 12
• 3) Glutamate decarboxylase 유전자 (gadA 및 gadB) 확보 및 염기서열 분석 15
• 4) 재조합 발현 벡터 구축 및 형질전환 17
• 5) 단백질 발현 및 SDS-PAGE에 의한 단백질 분석 18
• 6) Paper chromatography (PC) 분석 20
• 7) ABTS 이용한 GABA의 항산화 활성 측정 21
• 8) 동물세포 배양 및 세포 생존율 측정 (MTT assay) 22
• 9) Nitric oxide (NO) 생성량 측정 23
• 10) 통계적 분석 23
• 3. 결과 및 고찰 24
• 1) 대장균으로부터 glutamate decarboxylase 유전자 (gadA 및 gadB) 확보 24
• 2) 재조합 발현벡터 구축 35
• 3) 단백질 발현 유도 및 분석에 의한 발현 최적화 38
• 4) His-tag 도입에 의한 GadA 및 GadB의 항원항체반응 43
• (1) His-tag이 도입된 유전자 확보 43
• (2) pT7-7발현벡터로 gadA-His 및 gadB-His 유전자 도입 50
• (3) 단백질 발현 유도 및 분석 52
• 5) Paper Chromatography 분석 59
• 6) 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) 소거에 의한 항산화 활성 측정 64
• 7) E. coli 균주 GadA 및 GadB의 생산 및 부분정제 66
• 8) 당뇨세포주에서 MTT assay에 의한 세포 생존율 측정 68
• 9) Nitric oxide (NO) 생성량 측정 70
• 4. 요약 72
• Ⅲ. 실험 2. 고초균에서 유산균 균주로부터 유래된 글루탐산 탈탄산효소의 발현 및 분비: Gamma-amino butyric acid (GABA) 생성에 의한 생물학적 활성 76
• 1. 서론 77
• 2. 재료 및 방법 80
• 1) 재료 및 시약 80
• 2) Bacterial strains, plasmid 및 medium 81
• 3) GAD 유전자 확보 및 재조합 발현 분비벡터 구축 84
• 4) 고초균 형질전환 및 단백질 발현 분비 분석 85
• 5) Paper Chromatography (PC) 분석 86
• 6) 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) 소거 활성 측정(항산화활성) 87
• 7) 동물세포 배양 및 세포 생존율 측정 (MTT assay) 88
• 8) Nitric oxide (NO) 생성량 측정 89
• 9) 통계적 분석 89
• 3. 결과 및 고찰 90
• 1) 유산균으로부터 glutamate decarboxylase 유전자 (gadB) 확보 90
• 2) Glutamate decarboxylase 유전자 (gadB)를 포함하는 분비벡터 구축 92
• 3) 고초균 형질전환체서 GAD의 분비 발현 및 단백질 분석 97
• 4) 배양액에 분비된 GAD의 생물학적 활성 99
• (1) Paper chromatography에 의한 GABA 전환 99
• (2) ABTS+ 라디칼 소거에 의한 항산화 활성 측정 101
• (3) GABA 투여에 의한 당뇨세포주 생존복구율 및 nitric oxide생성량의 변화 103
• (4) GABA 투여에 의한 Nitric oxide (NO)의 변화 106
• 4. 요약 109
• Ⅳ. 인용문헌 111

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