초록 ▼

○ 연구결과
(1)반섬유소로부터 L-ribose의 생물학적 분해공정 개발
1.L-Arabinose생산:Endo-arabinanase와 alpha-L-arabinofuranosidase를 사용하여 반응조건을 최적화하여 최적 조건에서 20g/larabinan으로부터 2시간 동안 16g/lL-arabinose를 생산
2.반섬유소로부터 L-arabinose생산:볏짚 2g과 sugarbeet2g에서 각각 L-arabinose가 1.5mg과 12mg이 생산되어 sugarbeet가 8배 높은 효율을 보임.
3.L-Ribul

○ 연구결과
(1)반섬유소로부터 L-ribose의 생물학적 분해공정 개발
1.L-Arabinose생산:Endo-arabinanase와 alpha-L-arabinofuranosidase를 사용하여 반응조건을 최적화하여 최적 조건에서 20g/larabinan으로부터 2시간 동안 16g/lL-arabinose를 생산
2.반섬유소로부터 L-arabinose생산:볏짚 2g과 sugarbeet2g에서 각각 L-arabinose가 1.5mg과 12mg이 생산되어 sugarbeet가 8배 높은 효율을 보임.
3.L-Ribulose생산:L-Arabinoseisomerase를 사용하여 500g/lL-arabinose로부터 2시간 반응하여 95g/lL-ribulose생산
4. L-Ribose 생산: 효소의 유전자 진화와 분자모델링을 사용하여 효소를 개량하여 300 g/l L-ribulose로부터 1시간 동안 213g/lL-ribose를 생산
5. L-Arabinose로부터 L-ribose 생산 최적화: L-arabinose isomerase와 mannose-6-phosphate isomerase를 사용하여 500g/lL-arabinose으로부터 3시간 동안 118g/lL-ribose를 생산
(3)반섬유소로부터 L-arabinose및 L-ribose의 대량 생산 조건 확립
1.고정화 효소 함유 생물반응기에서 20 g/l sugar beet arabinan로부터 14일간 12 g/l이상 L-arabinose생산
2.고정화 효소 함유 생물반응기에서 300g/lL-arabinose로부터 10일간 50g/l이상 L-ribose생산
3.L-Arabinose및 L-ribose의 정제 조건 확립하고 시제품 L-ribulose19gram 및 L-ribose5.5 gram 제조

Abstract ▼

Ⅳ. Results
(1) Enzymatic hydrolysis of hemicellulose for L-arabinose production
1. Screening, selection, cloning, and expression of endo-arabinanases from Bacillus licheniformis and Caldicellulorsiruptor saccharolyticus; and alpha-L-arabinofuranosidase from C. saccharolyticus.
2. Enzyme pur

Ⅳ. Results
(1) Enzymatic hydrolysis of hemicellulose for L-arabinose production
1. Screening, selection, cloning, and expression of endo-arabinanases from Bacillus licheniformis and Caldicellulorsiruptor saccharolyticus; and alpha-L-arabinofuranosidase from C. saccharolyticus.
2. Enzyme purification, molecular mass determination, optimization of temperature and pH, thermostability measurement, and substrate specificity determination of endo-arabinanases from B. licheniformis and C. saccharolyticus; and alpha-L-arabinofuranosidase from C. saccharolyticus.
3. The ratio of two enzymes, temperature, pH, and the concentrations of enzymes and substrate were optimized. Under the optimal conditions, endo-arabinanase and alpha-L-arabinofuranosidase from C. saccharolyticus produced 16 g/l L-arabinose from 20 g/l arabinan.
4. L-Arabinose production from hemicellulose: 1.5 mg and 12 mg L-arabinose was produced 2 g rice straw and 2 g sugar beet, respectively.
(2) Bioconversion of L-arabinose into L-ribose
1. Screening, selection, cloning, and expression of L-arabinose isomerases from Geobacillus stearothermophilus, G. thermodenitrificans, and Thermotoga neopolitana; mannose-6-phosphate isomerases from Bacillus subtilus, G. thermodenitrificans, Thermus thermophilus, T. thermophilus R142N, and G. thermodenitrificans W17Q N90A L129F.
2. Enzyme purification, molecular mass determination, optimization of temperature and pH, thermostability measurement, and substrate specificity determination of L-arabinose isomerases from Geobacillus stearothermophilus, G. thermodenitrificans, and Thermotoga neopolitana; mannose-6-phosphate isomerases from Bacillus subtilus, G. thermodenitrificans, Thermus thermophilus, T. thermophilus R142N, and G. thermodenitrificans W17Q N90A L129F.
3. Optimization of L-ribulose production: The temperature, pH, and the concentrations of enzyme and substrate by L-arabinose isomerase from G. thermodenitrificans were optimized. Under the optimal conditions, the enzyme produced 95 g/l L-ribulose from 500 g/l L-arabinose for 2 h.
4. Optimization of L-ribose production: The temperature, pH, and the concentrations of enzyme and substrate by mannose-6-phosphate isomerases from B. subtilus, G. thermodenitrificans, and T. thermophilus were optimized. Under the optimal conditions, T. thermophilus mannose-6-phosphate isomerase produced 213 g/l L-ribose from 300 g/l L-ribulose for 3 h, with a conversion yield of 71% and productivity of 71 g L−1 h^-1.
5. mprovement for activities of L-ribose-producing enzymes by DNA evolution and molecular modeling
- A W17Q-N90A-L129F variant of G. thermodenitrificans mannose-6-phosphate isomerase was obtained using error-prone PCR and site-directed mutagenesis.
The specific activity, catalytic efficiency, and productivity of the variant were 3.1-, 7.1- ,and 4.5-fold higher than the wild-type enzyme, respectively. The variant produced 213 g/l L-ribose from 300 g/l L-ribulose for 1 h, with a conversion yield of 71% and productivity of 213 g L−1 h^-1.
- As L-ribulose was docked to T. thermophilus mannose-6-phosphate isomerase based on molecular modeling, its phosphate-binding site was found to be a molecular determinant. The R142N variant was developed through substrate-tailred optimization. The specific activity, catalytic efficiency, and productivity of the variant were 1.4-, 1.6-, and 1.5-fold higher than the wild-type enzyme, respectively. The variant produced 213 g/l L-ribose from 300g/l L-ribulose for 2 h, with a conversion yield of 71% and productivity of 107 g L−1 h^-1.
6. The ratio of two enzymes, temperature, pH, and the concentrations of enzymes and substrate were optimized. Under the optimal conditions, L-arabinose isomerase와 mannose-6-phosphate isomerase from G. thermodenitrificans produced 118 g/l L-ribose from 500 g/l L-arabinosefor 2 h, with a conversion yield of 24% and productivity of 39 g L−1 h^-1.
(3) Optimization for the production of L-arabinose and L-ribose from hemicellulose
1. Immobilized L-arabinose-producing enzymes in a bioreactor produced more than 12 g/l L-arabinose for 14 days from 20 g/l sugar beet arabinan at endo:exo ratio of 1:10 absorbed on Duolite A568, pH of 6.0, temperature of 80℃, and dilution rate of 0.6 h^-1.
2. Immobilized cells containing L-arabinose isomerase and mannose-6-phosphate isomerase in a bioreactor produced more than 50 g/l L-ribose for 10 days from 300 g/l L-arabinose absorbed on alginate at pH of 7.5, temperature of 60℃, and dilution rate of 0.1 h-1.
3. Purification of L-arabinose and L-ribose from the reaction products: After crystallization by adding 3 times volume of ethanol to the concentrated reaction solution, filtrate was applied to Dowex monosphere 99CA resin. The active fraction of the elute was collected, concentrated and dried. The dried product was the purified L-arabinose or L-ribose.
(4) Preparation of the purified L-arabinose and L-ribose as 19 gram of 97.5% L-ribulose and 5.5 gram of 80.9% L-ribose.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 6
• CONTENTS ... 9
• 목 차 ... 10
• 제 1 장 연구개발과제의 개요 ... 11
• 제 1 절 연구개발의 목적 ... 11
• 제 2 절 필요성 및 범위 ... 11
• 제 2 장 국내외 기술개발 현황 ... 13
• 제 1 절 시장 현황 ... 13
• 제 2 절 특허 현황 ... 15
• 제 3 절 논문 현황 ... 16
• 제 4 절 국내외 관련분야 환경변화 ... 16
• 제 3 장 연구개발수행 내용 및 결과 ... 18
• 제 1 절 반섬유소에서 L-arabinose 생산 ... 18
• 제 2 절 L-Arabinose에서 L-ribulose 생산 ... 35
• 제 3 절 L-Ribulose에서 L-ribose 생산 ... 41
• 제 4 절 L-Arabinose에서 L-ribose 생산 ... 61
• 제 5 절 L-Ribose 및 L-ribulose의 분리 정제 ... 78
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 83
• 제 1 절 연구개발 목표의 달성도 ... 83
• 제 2 절 정량적 목표의 달성도 ... 84
• 제 3 절 관련분야의 기술발전에의 기여도 ... 85
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 86
• 제 1 절 연구개발 성과 ... 86
• 제 2 절 성과활용 계획 ... 88
• 제 7 장 참고문헌 ... 91
• 끝페이지 ... 93