초록 ▼

○ 연구결과
인삼근권 토양으로부터 β-glucosidase 분비 미생물을 758개 screening하였고, 그 중에서 β-glucosidase 분비 능력을 재 조사하여 최종적으로 211개 균주를 확보하였으며, 전국 각지에서 지역별, 종류별로 104개의 김치시료를 수집하여 β-glucosidase 분비활성을 보이는 유산균 127개의 strain을 찾아내었다. 위의 미생물들 중에서 가장 β-glucosidase 분비활성이 뛰어나 Rg3로의 전환능력이 강한 Microbacterium esteraromaticum GS514를 분리하였

○ 연구결과
인삼근권 토양으로부터 β-glucosidase 분비 미생물을 758개 screening하였고, 그 중에서 β-glucosidase 분비 능력을 재 조사하여 최종적으로 211개 균주를 확보하였으며, 전국 각지에서 지역별, 종류별로 104개의 김치시료를 수집하여 β-glucosidase 분비활성을 보이는 유산균 127개의 strain을 찾아내었다. 위의 미생물들 중에서 가장 β-glucosidase 분비활성이 뛰어나 Rg3로의 전환능력이 강한 Microbacterium esteraromaticum GS514를 분리하였으며, 이 미생물에서 효소를 분리, 이를 이용하여 Rb1을 Rg3로의 전환을 할 수 있다는 결과를 얻어 내었다. 또한 이를 대량배양에 성공, 대량으로 Rg3 생산의 기반을 마련하였다. Rg3생산효소의 분리 동정은 세계적으로 처음으로 이루어지는 연구로서 학문적으로나 경제적으로나 모두 큰 가치를 가지게 되었다.

Abstract ▼

IV. Results and suggestions
1. Results
A. Enzyme types converting saponins
ß-glucosidase (sigma) was purchased commercially, and its ginsenoside Rb1-converting activity tested. 1U of ß-glucosidase and 1mM of ginsenoside Rb₁ were dissolved in 1.5 ml of phosphate buffer (50 mM, pH

IV. Results and suggestions
1. Results
A. Enzyme types converting saponins
ß-glucosidase (sigma) was purchased commercially, and its ginsenoside Rb1-converting activity tested. 1U of ß-glucosidase and 1mM of ginsenoside Rb₁ were dissolved in 1.5 ml of phosphate buffer (50 mM, pH 7.0). The reaction mixture was incubated for 24 hours at 30°C and analyzed by TLC, as below. Microorganisms were initially screened, using the Esculin-R2A agar, for their ability to produce ß-glucosidase. The black colonies on the Esculin-R2A agar that showed ß-glucosidase activity were picked and transferred tothe fresh Esculin-R2A agar. Pure cultures were checked for shape, color and size of colonies.
B. Screening of sponin-converting microorganisms
All the isolated strains were identified using their 16S rRNA gene sequences. 16S rRNA partial sequences, with sizes around 700 bp, were blasted in the NCBI database, with the closest type strains discovered. Most strains are classified as Gram-positive Actinobacteria, Bacilli and Proteobacteria. More precisely, the isolated ß-glucosidase-producing bacteria belonged to Streptomyces, Bacillus, Paenibacillus, Sphingomonas, Enterobacter and Escherichiagenera. The sequence similarity of their 16S rRNA genes to those of their closest type strains ranged from 96.0 to 100.0%. 8 major groups were found - Proteobacteria α-subdivision (28 isolates), Proteobacteria β-subdivision (10 isolates), Proteobacteria γ-subdivision (24 isolates), Actinobacteria (88 isolates), Firmicutes (28 isolates), Bacteroidetes (2 isolates), Deinococcus-Thermus (1 isolate) and Sphingopyrix granuli (1 isolate).
C. Selection of saponin-converting microorganisms
From ginseng rhizosphere, 211 β-glucosidase producing bacteria were isolated and from kimch, 111 bacteria were isolated. As a result of NMR analysis, the conversion products were ginsenoside Rd, 20(S)-Rg3, F2, compound K and gypenoside XVII.
Ginsenoside Rb1 was converted to Rd by Serratia fonticola FS6(2), Paenibacillus kobensis EM6(6)-a, Terrabacter tumescens DL6(1),Paenibacillus amylolyticus BB4(4)-b-1, Frateuria aurantia BB4(1)-b,Streptomyces bikiniensis BB5(7), Streptomyces galilaeus BB6(1),Streptomyces olivochromogenes BB6(2), Paenibacillus amylolyticus DB5(3),Sphingomonas echinoides BT4, Sphingomonas echinoides BT5,Sphingomonas echinoides BT9, Sphingomonas echinoides BT12,Sphingomonas echinoides BT14, Streptomyces tauricus BT5(6)-a,Cellulomonas uda T7-06, Cellulosimicrobium cellulans GS235, Terrabacter tumescens GS 462, Microbacterium esteraromaticum GS508, Microbacterium esteraromaticum GS514, Terracoccus luteus GS610, Streptomyces ferralitis GS614, Terrabacter tumescens GS653, Microbacterium esteraromaticum GS836, Microbacterium esteraromaticum GS844, Microbacterium resistens GS1184, Lactobacillus brevis KC-72, Lactobacillus sp. CS1 KC-102. Bacillus subtilis KC-103, Lactobacillus sp. KC-104, Lactobacillus sp. CS1 KC-105, Lactobacillus sp. CS1 KC-106, Lactobacillus sp. CS1 KC-107,Bacillus subtilis KC-150, Lactobacillus sp. CS1 KC-153 and Lactobacillus brevis KC-154.
Ginsenoside Rb1 was converted to Rg3 by Burkholderia stabilis EM3(2)-a, Terrabacter tumescens GS342, Microbacterium esteraromaticum GS508, Microbacterium esteraromaticum GS514, Terracoccus luteus GS610, Lactobacillus sakei KC-01, Leuconostoc pseudomesenteroides KC-02, Lactobacillus arizonensis KC-03, Leuconostoc mesenteroides KC-04, Lactobacillus arizonensis KC-09, Lactobacillus sakei KC-14, Lactobacillus plantarum KC-16, Lactobacillus brevis KC-19, Lactobacillus sakei KC-20, Lactobacillus sakei KC-21, Lactobacillus sakei KC-22, Leuconostoc mesenteroides KC-28, Lactobacillus brevis KC-37, Leuconostoc citreum KC-38, Leuconostoc mesenteroides KC-39, Weissella cibaria KC-40, Leuconostoc citreum KC-41, Lactobacillus sakei KC-43, Leuconostoc citreum KC-50, Lactobacillus sakei KC-51, Lactobacillus sakei KC-52, Lactobacillus sakei KC-53, Lactobacillus sakei KC-57, Lactobacillus sakei KC-58, Lactobacillus sakei KC-59, Lactobacillus sakei KC-60, Lactobacillus sakei KC-61, Leuconostoc citreum KC-62, Leuconostoc mesenteroides KC-63, Lactobacillus brevis KC-64, Lactobacillus plantarum KC-66, Lactobacillus plantarum KC-67, Lactobacillus plantarum KC-68, Lactobacillus plantarum KC-69, Lactobacillus plantarum KC-70, Lactobacillus arizonensis KC-78, Lactobacillus plantarum KC-79, Leuconostoc pseudomesenteroides KC-81, Weissella cibaria KC-82, Weissella cibaria KC-96, Weissella cibaria KC-97, Leuconostoc mesenteroides subsp. mesenteroides KC-100 and Lactobacillus plantarum KC-143
Ginsenoside Rb1 was converted to F2 by Paenibacillus kobensis EM6(6)-a, Frateuria aurantia BB4(1)-b, Sphingomonas yabuuchiae BT1, Caulobacter leidyia BT3, Sphingomonas echinoides BT9, Sphingomonas echinoides BT14, Streptomyces tauricus BT5(6)-a, Terrabacter tumescens GS462, Microbacterium esteraromaticum GS508, Terracoccus luteus GS603, Terracoccus luteus GS 608, Terracoccus luteus GS 610 and Lactobacillus plantarum KC-143.
Ginsenoside Rb1 was converted to compound K by Paenibacillus kobensis EM6(6)-a, Sphingomonas yabuuchiae BT1, Caulobacter leidyia BT3, Sphingomonas echinoides BT9, Sphingomonas echinoides BT14, Streptomyces tauricus BT5(6)-a and Terracoccus luteus GS610.
Ginsenoside Rb1 was converted to gypenoside XVII by Burkholderia stabilis EM3(2)-a, Terrabacter tumescens DL6(1), Frateuria aurantia BB4(1)-b, Streptomyces galilaeus BB6(1), Caulobacter leidyia BT3, Streptomyces tauricus BT5(6)-a, Terrabacter tumescens GS342, Terrabacter tumescens GS405, Terrabacter tumescens GS462, Terrabacter tumescens GS513, Streptomyces lienomycini GS516, Terracoccus luteus GS603, Terracoccus luteus GS608, Terracoccus luteus GS610 and Terrabacter tumescens GS653Y.
Major ginsenosides such as ginsenoside Rb1, Rb2, Rc and Rd can be easily converted into a mixture of 20(R)- and 20(S)-ginsenoside Rg3 by acid treatment or heating. But the isolation of each isomer from the racemic mixture is a time-consuming and complicated process. 20(S)-Rg3 has also been prepared by chemical synthesis from 20(S)-dammer-24-en-3α, 12β, 20-triol (betulafolienetriol), but the synthetic steps were complicated and the overall yield was low.
The enzymatic conversion through sugar hydrolysis at a specific position of ginsenoside is desirable for the production of 20(S)-Rg3. Until now no one has reported the production of ginsenoside Rg3 by using microbial enzymes. In this study, we isolated β-glucosidase-producing microorganism GS514 from soil around ginseng roots in a field using esculin-R2A agar, investigated the activity transforming ginsenoside Rb1 into Rg3, and identified its related metabolites.
D. Isolation and identification of Rg3-converting crude enzyme
Growth of the strain GS514 was very slow in nutrient broth and the activity producing Rg3 was very weak. In order to compare the effect of various media, the strain GS514 was cultured in LB broth, tryptic soy broth and nutrient broth for 24 h. Samples were collected at 6, 12, 18 and 24 h,and O.D was measured at 600 nm. The O.D of LB broth, tryptic soy broth and nutrient broth samples at 24 h were 2.346, 2.849 and 0.910, respectively. The growth rates in LB broth and tryptic soy broth were significantly higher than in the nutrient broth. Each suspension culture of the strain GS514 at 12 h was mixed with the same volume of ginsenoside Rb1 and then incubated for 10 h in a shaking incubator. We observed that cultures in LB broth and tryptic soy broth converted ginsenoside Rb1 into Rg3, but the culture innutrient broth only converted Rb1 to Rd. This suggested that LB broth and tryptic soy broth were suitable for the growth of the strain GS514 and also for production of Rg3. Therefore, LB broth, which costs low price than other media, was chosen for more study.
When the ginsenoside Rb1 was added to the culture broth of the strain GS514, the content of ginsenoside Rb1 and Rd gradually decreased and that of Rg3 gradually increased from 2 h to 8 h. This proved that metabolite Rd is a precursor of the ginsenoside Rg3. Similarly, when ginsenoside Rd was added to the culture broth of the strain GS514, the Rg3 was also produced and the content of the Rg3 remarkably decreased after 4 h. Conversion of Rd into Rg3 was faster than conversion of Rb1 into Rg3. This suggested that Rb1 was converted by different enzymes secreted by the strain GS514 in the following sequence: Rb1→d→g3. The enzymes hydrolyzed the terminal glucose and consecutively inner glucose at the C-20 position. Interestingly, after 10 h, the content of the ginsenoside Rg3 rapidly decreased. This may have occurred due to the decrement of the precursors (Rb1 and Rd) converted to ginsenoside Rg3 so the rate of production of Rg3 was slower than the rate of degradation of Rg3.
2. Suggestions
A. The Rg3 producing Microbacterium esterarmaticum GS514 will be applied to patent.
B. The Rg3 producing Microbacterium esterarmaticum GS514 can be used to produce the active enzyme in mass production.
C. After completing mass production, the production technique can be transferred to companies to produce functional food, medical product, etc.
D. The Rg3-converting enzyme was induced by inducer. Appling gene transformation technique, more cheap method for the production of Rg3 should be developed.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 11
• CONTENTS ... 19
• 목차 ... 23
• 제1장 연구개발과제의 개요 ... 27
• 제1절 연구개발의 목적 ... 27
• 제2절 연구개발의 필요성 ... 27
• 제3절 연구개발의 내용 및 범위 ... 30
• 제2장 국내외 기술개발 현황 ... 33
• 1. 국내·외 관련기술의 현황과 문제점 ... 33
• 2. 앞으로의 전망 ... 34
• 3. 기술도입의 타당성 ... 34
• 제3장 연구개발수행 내용 및 결과 ... 36
• 제1절 서론 ... 36
• 제2절 전환대상 사포닌 결정 및 사포닌 전환효소 type탐색 ... 37
• 1. 연구대상 사포닌 결정 ... 37
• 2. 효율적인 사포닌 추출방법 모색 ... 40
• 3. 사포닌전환효소 type탐색 ... 42
• 제3절 β-glucosidase 분비 미생물선별 ... 44
• 1. 토양에서 β-glucosidase 분비 미생물 분리 및 동정 ... 44
• 제 3 절 사포닌전환 미생물선별 ... 76
• 1. 재료 및 방법 ... 76
• 2. 결과 및 고찰 ... 85
• 가. 인삼사포닌 전환 반응시간 설정 ... 85
• 나. β-glucosidase분비 미생물에 의한 ginsenoside Rb1의 전환 TLC분석 결과 ... 85
• 다. β-glucosidase분비 미생물에 의한 ginsenoside Rb1의 전환산물 HPLC분석 결과 ... 98
• 라. NMR분석에 의한 Rb1전환산물의 구조동정 ... 107
• 제 4 절 인삼근권 토양으로부터 ginsenoside Rg3생산효소 분리 및 동정 ... 118
• 1. 재료 및 방법 ... 119
• 2. 결과 및 고찰 ... 128
• 가. Microbacterium esteraromaticum GS514균주에 의한 ginsenoside Rg3생산효소의 생산 최적조건 ... 128
• 나. Ginsenoside Rd, Rg3생산효소의 분리 ... 137
• 1). Ginsenoside Rb1으로부터 Rg3로의 전환 pathway 탐색 ... 137
• 2). Ginsenoside Rd생산효소 분리 ... 141
• 3). Ginsenoside Rg3생산효소 분리 ... 145
• 4). Ginsenoside Rg3생산효소를 이용한 ginsenoside Rb1전환율 측정 ... 157
• 제 5 절 GS 514 균주 조효소액에 의한 홍삼농축액, 조사포닌의 전환 및 사포닌 전환균주의 발효기 운전 ... 158
• 1. 재료 및 방법 ... 158
• 2. 결과 및 고찰 ... 159
• 가. Rg3생산조효소 대량생산 ... 159
• 나. Microbacterium esteraromaticum GS514 조효소액에 의한 홍삼농축액, 조사포닌의 전환 ... 160
• 나. 20L-fermenter를 이용한 발효기 운전 ... 168
• 제4장 목표달성도 및 관련분야에의 기여도 ... 175
• 제1절 연구개발 목표의 달성도 ... 175
• 제2절 관련분야의 기여도 ... 175
• 제5장 연구개발결과의 활용계획 ... 177
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 178
• 제7장 참고문헌 ... 178
• 끝페이지 ... 200