초록 ▼

○ 연구결과
페룰산을 대사하는 미생물들을 다수 분리하였으며, 이로부터 바닐린을 생산하는 세균 4주 및 방선균 2주를 확보하였다. 페룰산을 바닐린으로 전환시키는 유전자인 fcs와 ech를 Delftia acidovorans, Pseudomonas putida, Amycolaptosis sp. HR104로부터 클로닝 하였다. Streptomyces setonii의 genomic DNA library를 구축 및 난배양성 미생물로부터 신규 바닐린 생합성 유전자를 확보하기 위한 총 36종의 metagenome library의 확보하였다

○ 연구결과
페룰산을 대사하는 미생물들을 다수 분리하였으며, 이로부터 바닐린을 생산하는 세균 4주 및 방선균 2주를 확보하였다. 페룰산을 바닐린으로 전환시키는 유전자인 fcs와 ech를 Delftia acidovorans, Pseudomonas putida, Amycolaptosis sp. HR104로부터 클로닝 하였다. Streptomyces setonii의 genomic DNA library를 구축 및 난배양성 미생물로부터 신규 바닐린 생합성 유전자를 확보하기 위한 총 36종의 metagenome library의 확보하였다. 바닐린 유전자 (Amycolaptosis sp. HR104의 fcs와 ech)를 갖는 재조합 대장균에 페룰산, 1g/L를 기질로 첨가한 결과 바닐린, 약 0.5g/L가 생성되는 것을 확인하였다. 바닐린 생산 재조합 대장균에 gltA 유전자의 도입 등의 대사공학 기술을 적용해서 바닐린 생산성을 4배 이상 증가시켰다. 최종산물인 바닐린에 대한 내성이 약 2배 이상 증가한 돌연변이주를 확보하고 바닐린 생산성을 2배 이상 증가시켰다. 균체증식 및 바닐린 생산이 진행되는 발효조와 바닐린을 연속적으로 추출하는 추출조를 가지는 바닐린 생산용 신규 생물반응기를 개발해서 연속적인 바닐린의 생산을 가능하게 하였다.
페눌산 추출을 위해 11종의 목본류, 9종의 농업부산물을 추출하여 페눌산함량을 측정하였다. 이중 식용이면서 원료 확보가 용이한 고구마는 산가용성 함량이 18%, 산불용성 페놀함량이 0.95%로 나타났다. 고구마 줄기로부터 페룰산 분리에 적합한 조건을 구명하기 위해 산처리법, 알칼리처리법 및 고온고압처리 등을 이용하여 전처리법을 확립하였다. 그 결과 페놀성화합물의 최적 생산을 위해서는 전처리lignocellulose보다 고구마 줄기를 알칼리 처리하는 반응 조건이 유리하고, ferulic aicd 생산을 위해서는 고구마 줄기 원료보다는 전처리 lignocellulose 원료를 증류수만으로 단시간 고온 처리 (120℃) 가 유리하다고 판단되었다. 페룰산 순수단리를 위해 액체-액체 크로마토그라피법과 액체-고체 크로마토그라피법으로 적용하였다. 다단용제 중 페눌산은 ethyl acetate 분획에 약 13% 존재하는 것으로 나타났다. 또한 실리카겔을 충진한 컬럼크로마토그라피법에 의한 페눌산의 분리조건을 구명한 결과 n-hexane:ethyl acetate:formic acid = 80:50:0.5과 n-hexane:ethyl acetate:formic acid = 100:50:0.5 전개용매로 전개하였을 때 표준물의 분리가 가장 잘 되는 것으로 나타났다. 또한 vanillic acid와 ferulic acid의 분리도 양호한 수준으로 분리되었다. 최종적으로 전건상태의 바이오매스 원료 10 g 으로 부터 획득될 수 있는 ferulic acid의 중량은 고구마 줄기 원료는 54.1 mg 이었으며, 목질원료는 245.5 mg이었다.

Abstract ▼

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavors used in food, pharmaceuticals, and cosmetics industries. Natural vanillin is derived from the vanilla beans. Because of the climate-limited cultivation and labor-intensive process of vanilla flavor, vanillin is mainly of

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important flavors used in food, pharmaceuticals, and cosmetics industries. Natural vanillin is derived from the vanilla beans. Because of the climate-limited cultivation and labor-intensive process of vanilla flavor, vanillin is mainly of synthetic origin. In recent years, owing to the increasing demand for healthy and natural food, biovanillin produced from natural raw materials by metabolically engineered microorganism or plant tissue culture is expected to substitute for natural vanillin.
The most intensively studied process for producing vanillin by bioconversion, which then can be designated 'natural', is based on the substrate ferulic acld (4-hydroxy-3-methoxy-trans-cinnamic acid). Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and occurs mainly in cell walls covalently linked to lignin and other polymers. Several different pathways have beenproposed for the degradation of ferulic acid. The genes consisting of the CoA-dependent pathway, fcs and ech, encoding feruloyl-CoA-synthetase and enoyl-CoA-hydratase/aldolase respectively, can catalyze the conversion of ferulic acid to vanillin.
In our study, the fcs and ech genes were cloned from Amycolatopsis sp. HR104 by PCR, transferred to pBAD24 expression vector, and then resulted in pDAHEF. The recombinant E. coli harboring pDAHEF was used for vanillin production. In order to optimize induction condition of the recombinant E. coli, we investigated the concentration of inducer, arabinose and induction time on vanillin production. The optimum condition of induction was 0.2% arabinose induced at 18 hours of cultivation. Vanillin production was also maximized when 0.1% (w/v) ferulic acid was fed at induction point of 18 hours during cultivation, about
400mg/L of vanillin was obtained. Also, galactose as C-source and less complex media may assist the production of vanillin.
Both of the genes cloned from Amycolatopsis sp. HR104, were also transferred to pTrc99A expression vector, which resulted in pTAHEF. The recombinant E. coli harboring pTAHEF converted 0.1% and 0.2% ferulic acid to 650mg/L and 1200mg/Lof vanillin, respectively. Because vanillin is a highly toxic substance for most bacteria, it is possible to use some nonionic resins which enable vanillin to be trapped and its concentration to be maintained below the threshold of toxicity. Vanillin production increased with the use of XAD-2 resin, and 18.1g/L of ferulic acid was converted to 9.2g/L of vanillin using 10g XAD-2 resin under mimicking resting cell bioconversion. Citrate synthase gene gltA was also proved to increase the production of vanillin.
As mentioned above, vanillin is a highly toxic substance for most bacteria. There was a notable inhibition of E. coli at a vanillin concentration of 0.1% and the cell was no longer able to grow at concentrations higher than 0.25%. Therefore, we made the E. coli mutant which could grow at a vanillin concentration of 0.2% through NTG mutation increasing the tolerance of cells to vanillin. The highest yield of vanillin was obtained with the E. coli mutant harboring pTAHEF when it was cultivated for 48 hours in 2YT media containing ampicillin at 37℃ using shaking water bath with agitation of 180rpm. Under this cultivation condition, 1.94g/L of vanillin was produced from 0.3% ferulic acid.
While using the normal E. coli, only half amount of vanillin was produced from the 0.3% ferulic acid.
In order to minimize the toxicity of vanillin on its producing recombinant E. coli, a new bioreactor was developed. It is composed of two vessel for cell growth and continuos extraction of vanillin. When ferulic acid was supplied into the culture using the new bioreactor at concentration of 0.22% every 12 hours, 6.2g/L of vanillin was obtained.
In order to efficient extraction and purification of ferulic acid from agrocultural by-products including sweet potato tissues. Among agricultural by products and forest species that are potential sources of ferulic acid, sweet potato is probably one of promising, since it contains the high amount of phenypropanoid and ferulic acid. Chemical and physical treatment have been tested to improve ferulic acid release.
Qercus variabilis was possess of most abundant of acid insoluble and acid soluble phenylpropanoid compound among the selected hardwood species. Q. variabilis was suitable for this study, because this species was easy of supply in side of actuality.
Vanillin was final product of this study, and to production of vanillin, sweet potato stem thought to the most suitable source because of edible resource.
However, amount of phenylpropanoid compound from sweet potato stem was lower than other agricultural by-products. But in Korea, food- and drug-related products or permission and registration of the materials were necessarily limited in the resources which is eatable. But, contents of phenylpropanoid compound from agricultural by-products such as tomato or sesame stem were consisted over 25% of total raw material weight.
Both Extract yield and phenylpropanoid compound yield was risen from sulfuric acid addition reaction, but clear relation was not appear between both yield from acetic acid addition reaction. From the condition which does not consider an economic problem, it was decided that using the alkali for 90 min at room temperature was most profitable. At this time, the yield of phenylpropanoid compound was 403.5 mg, the value was approximately 40% when this changes into dry weight basis of input materials.
Also when using the alkali was react at 120℃, yield of phenylpropanoid compound was higher, approximately 2.5-fold, than reaction by acid. Finally, to optimal production, the condition of alkali treated reaction from sweet potato stem was profitable than pre-treated lignocellulose, and to production of ferulic acid, it was decided that pre-treated lignocellulose material used only distilled water in short time high temperature (120℃) was profitable than sweet potato stem.
In this results, it required about ￦20,000 won for production of 1g ferulic acid from pre-treated lignocellulose. The method with material as pre-treated lignocellulose was reduced approximately 50% of expenditure than production of ferulic acid from sweet potato setm. Most of the cost was reactor which constituted electric charges of room temperature stirrer equipment and collection equipment. Therefore it is thought that scale up is enough to economical efficiency.
Finally, most of phenypropanoid compound was 95% exceed the ethyl acetate insoluble fraction. To separate and purify of ferulic acid which is manufacturing material of bio vanillin from sweet potato stem and woody material one of the agricultural and forest product biomass materials, liquid-liquid chromatography and liquid-solid chromatography technology was applied however, it could not get a satisfied results. Thus, in this section, multi-solvent extraction (liquid-liquid chromatography) and silica-gel column chromatography (liquid-solid chromatography) were consecutively applied to separate of ferulic acid.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 6
• CONTENTS ... 10
• 목차 ... 13
• 제 1 장 연구개발과제의 개요 ... 16
• 제 1 절 연구개발의 목적 ... 16
• 1. 최종목적 ... 16
• 2. 단계별 목표 ... 16
• 제 2 절 연구개발의 필요성 ... 16
• 1. 기술적 측면 ... 16
• 2. 경제·산업적 측면 ... 21
• 3. 사회·문화적 측면 ... 23
• 제 2 장 국내외 기술개발현황 ... 24
• 제 1 절 천연바닐린의 생산 ... 24
• 1. 재배에 의한 바닐린 생산 ... 24
• 2. 바닐라 꼬투리 가공 ... 27
• 3. 용매추출 ... 27
• 제 2 절 화학합성 바닐린의 생산 ... 27
• 1. Coniferin으로부터 바닐린 생산 ... 28
• 2. Eugenol로부터 바닐린 생산 ... 28
• 3. Lignin으로부터 바닐린 생산 ... 28
• 4. Guaiacol로부터 바닐린 생산 ... 28
• 제 3 절 바이오 바닐린의 생산 ... 29
• 1. 식물세포배양에 의한 바닐린 전구체의 생물전환 ... 32
• 2. 미생물 배양에 의한 바닐린 전구체의 생물전환 ... 32
• 3. 바닐린 대사경로의 유전자원 및 대사공학에의 응용 ... 32
• 4. 페룰산 대사능과 바닐린 내성이 큰 돌연변이주의 개발 ... 33
• 5. 고 생산성 바닐린 생산공정 개발 연구 ... 33
• 제 4 절 국내 관련기술 ... 35
• 제 5 절 앞으로 전망 ... 35
• 제 6 절 기술도입의 타당성 ... 36
• 제 3 장 연구개발수행 내용 및 결과 ... 37
• 제 1 절 연구개발 목표와 내용 ... 37
• 1. 총괄목표 ... 37
• 2. 세부과제별 목표 및 내용 ... 37
• 제 2 절 연차별 연구개발 목표와 내용 ... 38
• 제 3 적 연구개발 및 설계 ... 41
• 1. 제 1 세부과제 (대사공학 기술을 이용한 우수 바닐린생산균주 개발) ... 41
• 2. 제 2 세부과제 (Lignocellulosic 원료로부터 바닐린 전구체 생산기술개발) ... 47
• 제 4 절 연구개발 추진체계 ... 60
• 제 5 절 연구수행 결과 ... 61
• 1. 제 1세부과제 (대사공학 기술을 이용한 우수 바닐린생산균주 개발) ... 61
• 2. 제 2 세부과제 (Lignocellulosic 원료로부터 바닐린 전구체 생산기술개발) ... 111
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 156
• 제 1 절 연구평가의 착안점 ... 156
• 제 2 절 연구개발 목표 측면에서의 달성도 평가 ... 157
• 1. 제 1 세부과제: 대사공학 기술을 통한 바닐린 생산균주의 생산성 개량 (목표대비 100% 달성) ... 157
• 2. 제 2세부과제: Lignocellulose 원료로부터 바닐린 전구체 생산기술 개발 (목표대비 100% 달성) ... 159
• 제 3 절 관련분야 및 기술발전에의 기여도 ... 161
• 1. 기술적 측면 ... 161
• 2. 경제·산업적 측면 ... 161
• 제 5 장 연구개발결과의 활용계획 ... 163
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 164
• 제 7 장 창고문헌 ... 165
• 끝페이지 ... 173