Optimization of lipase-catalyzed synthesis of chlorogenate ester

綠原酸(chlorogenic acid)可被當為有機相中之抗氧化劑，但綠原酸在有機相的溶解度較差，因此提高綠原酸的親脂性為本文研究的方向。 本實驗分成兩部分 第一部分以綠原酸當成酸和辛醇(ocatanol)來進行酯化反，應生成綠原酸酯，實驗探討反應時間(12h ~ 36h)、酵素用量(10 ~ 50mg)及超音波功率(90 ~ 150W)對綠原酸轉化率之影響，結果顯示反應時間、酵素用量對於綠原酸的轉化率皆有顯著影響。以三階層三變數之 Box-Behnken design (BBD)及反應曲面法探討綠原酸酯化之轉化率，結果顯示最適化合成反應條件為反應時間12h，酵素用量 50mg 及功率 120W，此條件下所得到綠原酸轉化率為 95.3%。 第二部分以綠原酸當醇並和辛酸(octanoic acid)進行反應並以固定化酵素Novozyme 435 催化合成綠原酸酯，在使用溶劑 2M2B 情況下進行合成。實驗選取三個反應因素因子莫爾數比 (綠原酸比辛酸)、酵素用量、反應時間。其中莫爾數比及酵素用量皆會對轉換率產生極大影響。再以三階層三變數之 Box-Behnken design (BBD)及反應曲面法探討綠原酸酯化之轉化率，分析對合成綠原酸酯的影響。實驗結果最適化條件為：莫爾數比 1:300、酵素用量 140mg 及反應天數為 2 天時，可得到綠原酸酯轉化率為 36.5%。 關鍵字:脂解酵素、反應曲面法、綠原酸、最適化、酯化反應、酵素合成Chlorogenic acid is usually used as antioxidants in the organic phase; however, its low solubility causes a problem when applied in industry. To increase the lipophilic property of chlorogenic acid is the primary aim in this study. This study is divided into two parts. In the first part, chlorogenic acid is used as an acid and octanol was employed as alcohol in the esterification reaction to produce chlorogenic acid ester. Factors such as reaction time (12-36 h), enzyme amount (10-50 mg) and power (90-150 W) were studied. The results show that the reaction time and the enzyme amount have the most significant effect on the conversion of chlorogenic acid. The 3-level-3-factor Box-Behnken design (BBD) was applied to optimize the reaction conversion. The optimal reaction conditions are as follows: reaction time 12 h, enzyme amount 50mg, and ultrasound power of 120W. Under this condition, an esterification conversion of 95.3% can be reached. In part two, chlorogenic acid is employed as an alcohol and octanoic acid was used as the acid in the esterification reaction to produce chlorogenic acid ester. 2-methyl-2-butanol (2M2B) is chosen as the solvent. Box-Behnken design was adopted to evaluate the effect of three different parameters, i.e., molar ratio (1:100-1:300), enzyme amount (20-140mg) and reaction time (1-3day). The results show that the molar ratio and the enzyme amount have the most significant effect on chlorogenic acid conversion. By ridge max analysis, the optimal reaction conditions were found as follows: molar ratio 1:348.5, enzyme amount 159.6 mg, and reaction time 2.19 days. Under this condition, the highest conversion of 36.8%±0.8 can be obtained. Keywords: Lipase、response surface methodology、chlorogenic acid、optimal、esterification、enzymatic synthesis第一章緒論................................................. 1 1.1 前言.................................................. 1 1.2 研究動機.............................................. 2 第二章文獻回顧............................................. 3 2.1 綠原酸及綠原酸酯的介紹................................. 3 2.1.1 綠原酸.............................................. 3 2.1.2 綠原酸酯............................................ 4 2.2 綠原酸酯的合成........................................ 5 2.2.1 酵素介紹............................................ 5 2.2.2 脂肪分解酵素(Lipase)介紹 ............................ 5 2.2.3 Candida antarctica lipase-B (CALB) ................. 6 2.3 固定化酵素介紹......................................... 7 2.3.1 固定化酵素起源....................................... 7 2.3.2 固定化酵素種類....................................... 7 2.3.3 固定化酵素之優點..................................... 8 2.4 相關研究............................................. 10 2.5 不同有機溶劑中之酵素反應.............................. 11 2.6 以超音波進行酯化酚酸之研究............................. 14 2.6.1 超音波的基本性質.................................... 14 2.6.2 超音波種類.......................................... 14 2.6.3 相關超音波輔助合成研究.............................. 16 2.7 最適化分析............................................ 18 2.7.1 單因子實驗法........................................ 18 2.7.2 反應曲面法.......................................... 18 2.7.3 全因子實驗設計(Full factorial design) .............. 19 2.7.4 Box-Behnken 實驗設計(Box-Behnken design, BBD) ..... 20 2.8 總結................................................. 20 第三章材料與方法.......................................... 21 3.1 實驗流程............................................. 21 3.1.1 以綠原酸(CQA)當酸 .................................. 21 3.1.2 以綠原酸當醇........................................ 22 3.2 實驗藥品............................................. 23 3.3 實驗儀器............................................. 24 3.4 實驗方法............................................. 25 3.4.1 分析條件........................................... 25 3.4.2 綠原酸酯產率計算.................................... 25 3.4.3 統計分析........................................... 26 3.4.4 以超音波輔助合成綠原酸酯............................ 27 3.4.4.1 實驗的基本介紹.................................... 27 3.4.4.2 合成綠原酸酯的步驟................................ 28 第四章 以綠原酸當酸進行合成之結果與討論.................... 29 4.1 圖譜分析............................................. 29 4.2 單一因子最適化探討.................................... 30 4.2.1 反應時間........................................... 30 4.2.2 酵素用量........................................... 31 4.2.3 功率探討........................................... 32 4.3 反應曲面法設計........................................ 33 4.4 合成綠原酸酯之變數分析................................ 35 4.5 反應曲面分析.......................................... 37 4.6 反應曲面法穩定性...................................... 41 第五章 以綠原酸當醇進行合成之結果與討論(PART 2 ) .......... 42 5.1 圖譜分析............................................. 42 5.2 單一因子最適化探討.................................... 43 5.2.1 莫爾數比........................................... 43 5.2.2 酵素用量........................................... 44 5.2.3 反應天數探討........................................ 45 5.3 反應曲面設計......................................... 46 5.3 合成綠原酸酯之變數分析................................ 48 5.4 反應曲面分析.......................................... 50 第六章結論與未來展望...................................... 55 6.1 結論................................................. 55 6.2 未來展望............................................. 5