脂肪酶催化果醣脂肪酸酯的合成及應用

Abstract

脂肪酸糖酯是無毒、無味、無刺激性的界面活性劑。它們可以由再生資源生產，並且可以完全被生物降解。脂肪酸糖酯的應用已擴展到許多領域，包括製藥、化妝品、洗滌劑和食品工業。果糖是一種存在許多植物中的單糖酮醣，它通常與葡萄糖結合形成雙醣蔗糖。過去，本實驗室發現果糖具有抗內毒素和抗氧化的功能，這開啟了對果糖新的潛在功效和應用的研究。為了增加對內毒素的親和力，應增加果糖的疏水性。在本研究中，我們利用市售的固定化脂肪酶Novozymes 435催化脂肪酸酯和果糖之間的轉酯化反應，從而生成果糖酯。藉由MPLC和HPLC分析可獲得果糖轉化產率(69.2%)和各個果糖酯的產率(單丙酸果糖酯-1，即FMP1為23.4%、單丙酸果糖酯-2，即FMP2為21.6%、雙丙酸果糖酯，即FDP為29.6%、單月桂酸果糖酯，即FML2為24.2%)。這些果糖酯吸附內毒素和清除自由基的能力也已經透過生化分析進行。除此之外，在活體試驗中，我們也利用內毒素誘導Wistar雌性大鼠導致敗血症之模型，觀察果糖酯是否能保護模式老鼠，減輕因內毒素而引起的症狀，包括對組織血流量變化與損傷影響，此外也利用西方墨點法（western blotting）去進一步的觀察內毒素增加PARP、4HNE及caspase 3等細胞凋亡相關蛋白的變化。而果糖酯可以降低細胞凋亡蛋白的表現，證明果糖酯的保護效應。因此，本研究建立了果糖酯的酶合成法以用於工業應用，開發果糖酯的新型生化活性以用於醫藥、醫療器材和保健食品工業。Fatty acid sugar esters are non-toxic, odorless, non-irritating surfactants. They can be produced from renewable resources and are fully biodegradable. Their applications have been extended in many areas including the pharmaceutical, cosmetic, detergent and food industries. Fructose is a simple ketonic monosaccharide found in many plants, where it is often bonded to glucose to form the disaccharide sucrose. Previously, our group has found that fructose can be an anti-endotoxin and anti-oxidant agent, and this opens the investigation on the novel potential efficacy and the application of fructose. In order to increase the affinity toward endotoxin, the hydrophobicity of fructose should be increased. In this study, Novozymes 435, a commercial immobilized lipase, was used to catalyze the transesterification between fatty acid ester and fructose to produce fructose esters. The fructose conversion rate and the individual fructose esters yields have been analyzed by MPLC and HPLC. The endotoxin and free radical removal capacity of these synthesized fructose esters have also been carried out by biochemical assays. In addition, in in vivo experiments, to determine whether fructose esters have protective effects, we treated Wistar female rats with various fructose esters before inducing septicemia with endotoxin. Flow rate change of blood stream and tissue damage level will be analyzed. Western blotting is also be performed to observe the effects on the cell apoptosis related proteins including PARP, 4HNE and caspase 3. Therefore, the results of this study are set up an enzymatic production process of fructose esters for the industrial application, and exploit the novel biochemical activity of fructose esters for the applications in medicine, medical devices and health food industries.