유기용매계에서 lipase-촉매 에스테르화 반응에 의한 erythorbyl laurate의 합성 : Lipase-catalyzed Esterification in Organic Medium for the Synthesis of Erythorbyl Laurate

국문 초록 (Abstract)

Erythorbic acid는 ascorbic acid(Vit. C)의 입체 이성질체(stereoisomer)로서, 항산화제로 널리 사용되어 왔으나 지용성이 낮은 특성으로 인해 지질식품에는 적용의 한계가 있었다. 이러한 문제를 극복하기위해, erythorbic acid와 lauric acid를 기질로 lipase-촉매 esterification 반응을 유기용매계에서 수행하였다. 생성물인 erythorbyl laurate와 기질은 HPLC를 이용하여 정량적으로 분석하였으며, erythorbic acid, lauric acid 및 erythorbyl laurate의 머무름시간(retention time)은 각각 2.506±0.014, 3.386±0.027 및 4.628±0.032 분으로 확인되었다. 반응수율의 극대화를 위해, 효소적 에스테르화 반응의 경시적 변화 모니터링 및 유효한 반응변수(효소 농도, 기질의 조성비 및 반응온도)가 효소적 에스테르화 반응에 미치는 영향에 대한 연구를 수행하였다. 그 결과로서, 효소적 에스테르화 8시간 후 반응은 평형상태(equilibrium state)에 도달하였으며, 효소적 에스테르화 반응을 위한 효소 농도, 기질의 조성비 및 반응온도의 최적조건은 각각 20 mg solid/mL, 10([lauric acid]/[erythorbic acid]) 및 50℃로 나타났다. 효소적 에르테르화 반응에 의해 합성된 생성물은 분리정제 과정을 거친 후, LC-ESI, 1H- 및 13C-NMR를 이용한 구조분석을 통해 erythorbyl laurate(6-O-lauroyl-erythorbic acid)로 규명되었다. 본 연구의 결과로부터, 효소적 에스테르화 반응에 의해 생산된 erythorbyl laurate는 유화제 및 지질식품에 적용 가능한 항산화제로서 사용할 수 있을 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Erythorbic acid, a stereoisomer of ascorbic acid, has been used as an antioxidant, but limited in application to lipid by reason of its poor lipophilicity. To overcome this problem, lipase-catalyzed esterification between erythorbic acid and lauric acid was performed in acetonitrile. Substrates and erythorbyl laurate produced were analyzed quantitatively using HPLC and the retention times of erythorbic acid, lauric acid, and erythorbyl laurate were determined as 2.506±0.014, 3.386±0.027, and 4.628±0.032 min, respectively. Time course of the enzymatic esterification was monitored and effect of reaction variables, such as lipase content, the molar ratio of lauric acid to erythorbic acid ([lauric acid]/[erythorbic acid]), and reaction temperature on the enzymatic reaction were investigated to increase yield of molar conversion. From the results, a steady state was achieved after 8 hr from the initiation of the esterification and optimum conditions of lipase content, the ratio of lauric acid to erythorbic acid, and reaction temperature were 10 mg solid/mL of reactor, 10 ([lauric acid]/[erythorbic acid]), and 50℃, respectively. After purification procedure, the product synthesized by the immobilized lipase (Novozym？？435) was identified to be erythorbyl laurate (6-O-lauroyl-erythorbic acid) from LC-ESI-MS, 1H-, and 13C-NMR structural analysis. The results suggest that erythorbyl laurate produced in this study could be used as a new kind of emulsifier with antioxidative activity to retard oxidation of lipid-based foods.

목차 (Table of Contents)

• Abstract ⅰ
• List of Figures ⅲ
• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 3
• 1. Materials 3
• 2. Procedure for the lipase-catalyzed esterification 3
• 3. HPLC analysis 4
• 4. urification and structural analysis of erythorbyl laurate by LC-ESI-MS, 1H-, and 13C-NMR 5
• Ⅲ. Results and Discussion 6
• 1. Time course of enzymatic synthesis of erythorbyl laurate in acetonitrile 6
• 2. Optimization of conditions for the esterification catalyzed by immobilized lipase from Candida antarctica 12
• 3. Structural analysis of erythorbyl laurate synthesized through lipase-catalyzed esterification 16
• Ⅳ. Conclusion 19
• References 20
• Summary (in Korean) 23
• Appendix (abbreviation) 24