초록

○ 연구결과
① 국내산 배추로부터 PLD 추출, 정제에 관한 효과적 생산 기술을 개발하여 7,500Unit/kg(배추)의 효소를 추출하고, 이를 재사용 하기 위한 고정화 기술 개발
② 배추 유례 PLD와 난황유래 레시틴의 효소반응을 통해 PS를 합성하고 정제하여 60% 이상 순도의 PS 생산 기술 개발
③ DHA 를30% 이상 포함하는 인지질 생산 기술을 개발 하고, PLA2 고정화를 통한 재사용 기술 개발

Abstract ▼

Ⅳ. Result
1. Production of PL-PUFA
Experiments under various conditions to optimize the interesterification reaction of phospholipid and PUFA(polyunsaturated fatty acids) were made. As a result phospholipid containing 30% of PUFA was synthesized. And non-hydrolyzed phospholipid into lysophosph

Ⅳ. Result
1. Production of PL-PUFA
Experiments under various conditions to optimize the interesterification reaction of phospholipid and PUFA(polyunsaturated fatty acids) were made. As a result phospholipid containing 30% of PUFA was synthesized. And non-hydrolyzed phospholipid into lysophospholipid was recovered as much as 67%. When it comes to immobilized Lipozyme(TM), it was easily separated from the substrate, and showed relatively high incorporation rate of PUFA and phospholipid recovery yield compared to Lecitase(TM). Highly purified phospholipid up to 98% was recovered by supercritical carbon dioxide extraction method in 60 min to separate the free fatty acids from phospholipids.
2. Purification and characterization of chinese cabbage
Phospholipase D(PLD) from chinese cabbage was purified with mild and nontoxic process. Scalable purification method to mass production was established by combining filtration, precipitation, centrifugation. Multiple cascading filtration steps with chilling below ambient temperature were performed. Precipitation of the enzyme was induced by heating, organic solvent, and salt and subsequent centrifugation process was performed.
The established process combining various relatively easy methods to extract and separate PLD was practical and effective.
The specific activity of the first chinese cabbage PLD was around 30~40 unit/mg, the stability of which was enhanced in the eggyolk lecithin(GL-90E) solution mostly consisting of egg phosphatidylcholine. Hence it was possible to react for 48 hours and more at 50 . As a result, industrial ℃ application of phosphatidylserince synthesis became promising.
3. Enzyme immobilization (PLA2, PLD)
Immobilized enzyme with covalent binding method maintaineed 80% of the initial activity for 200 hour usage, and alginate-silicate sol-gel matrix method showed great stability to be used in industrial production by maintaining 80% of the initial activity. Especially, sol-gel matrix method can be implemented with easy and cheap production process and showed several advantages to be applied in commercialization.
Production efficiency and activity were enhanced by the method in which enzyme crosslinking and covalent binding onto the surface of silica gel were implemented simultaneously. PLA was immobilized using glutaraldehyde and silica gel, and the immobilized enzyme maintained the activity of liquid state. PLD was immobilized using the same method as well, and its activity varied more delicately bydifferent amount of crosslinking agent, but the appropriate level of dosage was found after all.
4. PS production - with cabbage PLD
Transphosphatidylation using PC as a substrate with commercial cabbage PLD was performed. To avoid toxicity of organic solvent, reaction experiments were performed in aqueous system. After optimizing reaction condition, over 40% of PS(phosphatidylserine) was synthesized and reproducible reaction method with over 90% of phospholipid recovery was established. Analysis method of phospholipid profile with HPLC was established and stabilized.
5. PS production - with chinese cabbage PLD
After experimental trials to optimize the reaction condition with the inhouse chinese cabbage PLD, PS synthesis was increased up to 54% using egg yolk lecithin(GL-90E) produced by Goshenbiotech. Various reaction conditions were screened and enzyme activity, stability were analyzed and subsequent analysis was made for the necessary factors for scaling up to commercial plant. These factors include substrate concentration, reaction temperature, reaction time, agitation state. Through the experimental screening process, optimized condition of PS synthesis was found by balancing factors such as substrate species and purity, pH value, reaction time, temperature, surfactant. And comparison of ps synthesis condition between conventional cabbage PLD and domestic chinese cabbage PLD was made. By taking into account of this finding, PS was synthesized up to 53.6% from not a pure PC but a GL-90E, and this shows greater conversion activity of chinese cabbage PLD than cabbage PLD.
6. PS purification
Salting out method with NaCl and two phase aqueous extraction with ethyl acetate were applied to purify the PS. The result showed that it is possible to easily isolate PC with residual PA and enhance the purity of PS.

목차 Contents

• 제출문 ... 1
• 보고서 요약서 ... 2
• 요약문 ... 3
• SUMMARY ... 8
• Contents ... 15
• 목차 ... 16
• 제 1 장 연구개발과제의 개요 ... 17
• 제 1 절 연구개발과제의 목적 ... 17
• 제 2 절 연구개발과제의 필요성 및 범위 ... 18
• 제 2 장 국내외 기술개발 현황 ... 21
• 제 1 절 PL-PUFA 생산 ... 21
• 제 2 절 PLD 정제 및 특성 ... 22
• 제 3 절 효소 고정화 ... 23
• 제 4 절 PS 생산 ... 24
• 제 3 장 연구개발수행 내용 및 결과 ... 25
• 제 1 절 PL-PUFA 생산 ... 25
• 제 2 절 PLD 정제 및 특성 ... 60
• 제 3 절 효소 고정화 (PLA2, PLD) ... 86
• 제 4 절 PS 생산 - 양배추 PLD 이용 ... 109
• 제 5 절 PS 생산 - 국산배추 PLD를 이용한 PS 생산 ... 120
• 제 6 절 PS 정제 - 인지질 혼합물로부터의 PS정제 ... 128
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 133
• 제 1 절 연구개발의 목표 및 평가의 착안점 ... 134
• 제 2 절 연구개발 수행내용 및 목표달성도 ... 135
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 136
• 제 1 절 실용화 및 산업화 계획 ... 136
• 제 2 절 기술 확산 계획 ... 137
• 제 3 절 지적 재산권 확보 계획 ... 137
• 제 4 절 추가 연구 및 타 연구 활용 계획 ... 138
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 139
• 제 7 장 참고문헌 ... 140
• 끝페이지 ... 144