HPLC多相酶促合成咖啡酸苯乙酯的工艺设计及微反应器强化
建立了咖啡酸苯乙酯和咖啡酸烷基酯的HPLC分析方法，制备了合成底物咖啡酸甲酯。以咖啡酸甲酯和苯乙醇作为反应底物，通过多相酶促反应合成了咖啡酸苯乙酯。反应介质采用等体积的离子液体[Bmim][Tf2N]和含络合剂（三甲基氧化磷）25%的环己烷，以固定化脂肪酶Novozym 435为催化剂，当反应温度为75 ℃、咖啡酸甲酯和苯乙醇的摩尔比为1:20、反应时间为60 h时，咖啡酸苯乙酯的得率高达89。3%。
使用HPLC分析方法检测蜂胶的成分，成分有咖啡酸甲酯、咖啡酸乙酯、咖啡酸丙酯、咖啡酸丁酯、咖啡酸戊酯及咖啡酸苯乙酯等。以咖啡酸酯提取物替代咖啡酸甲酯为底物，提取物与苯乙醇的底物比是1:15，在相同的反应介质和催化剂条件下，反应48 h的得率为74。2%。
应用微反应器，以咖啡酸甲酯和苯乙醇作为反应底物，通过多相酶促反应合成了咖啡酸苯乙酯，研究流速和温度对得率的影响。结果表明，当底物浓度为3 g/L、咖啡酸甲酯和苯乙醇的摩尔比为1:20、反应温度为65 ℃、微流的流速为2 μL/min时，反应2。5 h的得率为67。9%，比常规反应器下的反应时间缩短为1/24。
咖啡酸苯乙酯对青枯菌抑菌效果的研究，抑菌圈实验证明了咖啡酸苯乙酯对青枯菌的生长有很好的抑制效果。当咖啡酸苯乙酯的浓度为2。0 g/L时，培养24 h时的抑菌效果最高，达64。3%；当咖啡酸苯乙酯的浓度为1。0 g/L，培养48 h时，抑菌效果最高可达67。3%。
毕业论文关键词：多相反应体系；咖啡酸苯乙酯；咖啡酸酯提取物；微反应器；青枯菌
Abstract Caffeic acid phenethyl ester (CAPE) is a main ingredient of propolis。 It has the antioxidant, anti-inflammatory, and anti-tumor activity。 Because of the low yield and various by-products after extracting and chemical synthesizing methods, enzymatic synthesizing CAPE has become the research hotspot。 In this paper, enzymatic synthesis process of CAPE with multiphase was conducted, and strengthened the technological process by microreactor, and preliminarily discussed the antibacterial activity of CAPE。
HPLC analysis methods of CAPE and caffeic acid ester extract were established, and the substrate MC was prepared。 CAPE was synthesized by multiphase enzymatic pathways with the substrates of MC and phenyl ethanol。 CAPE yield was obtained of 89。3 % with the conditions of Ionic liquid [Bmim][Tf2N], complexing agent reaction as multiphase reaction medium, catalyst of Novozym 435, 75 oC of reaction temperature, 1:20 of substrate molar ratio, 60 h of reaction time。
HPLC analysis method was used to detect the components of propolis, including MC, EC, AC, PC, BC and CAPE。 CAPE yield was obtained of 74。2 % with conditions of MC as the substrate, 1:15 of the substrate ratio, 48 h of reaction time under the condition of the same reaction medium and catalyst。
CAPE was synthesized by multiphase enzymatic pathways in microreactor with the substrates of MC and benzyl alcohol, and the factors of flow rate and temperature were studied。 CAPE yield was obtained of 67。9 % in 2。5 h with the conditions of 3 g/L substrate concentration, 1:15 molar ratio of coffee acid methyl ester and benzene ethanol, 65 oC of reaction temperature, 2 μL/min of flow rate。 Compared with the common reactor, the reaction time is reduced to 1/24。
The antibacterial effect of CAPE on Ralstonia solanacearum was studied。 The inhibitory effect of CAPE on the growth of Ralstonia solanacearum was proved by inhibition zone。 The highest inhibition rate was obtained of 64。3 % by culturing 24 h when the concentration of CAPE was 2。0 g/L。 When CAPE concentration was set as 1 g/L, the inhibition rate was obtained the maximum of 67。3 % by culturing 48 h。