【作者】 陈瑜；

【导师】 黄锡荣；

【作者基本信息】 山东大学 ， 分析化学， 2025， 硕士

【摘要】 脂肪酶是重要的酶制剂,在手性芳基醇/酸的选择性制备领域具有重要的意义,为实现亲水性脂肪酶与疏水性底物的介质兼容,微乳液常用作脂肪酶促反应的介质。微乳液介质有多种类型,通过调控微乳液的组成可以构建出适用于脂肪酶催促不同反应的微乳液介质,为手性芳基醇/酸的高效生物制备提供实验支撑。为此,本文开展了如下两个方向的研究工作:1、CHAPS提高AOT反胶束中脂肪酶催化(S)-布洛芬正辛酯选择性合成反应效率的研究本文探究了生物兼容面型两性离子表面活性剂CHAPS(3-[(3-胆酰胺丙基)二甲氨基]丙磺酸内盐)对AOT(二(2-乙基己基)琥珀酸酯磺酸钠)反胶束体系微结构及增溶脂肪酶催化(S)-布洛芬正辛酯选择性合成反应的影响。结果表明,CHAPS可以提高AOT反胶束中脂肪酶催化(S)-布洛芬正辛酯合成反应的产率和对映体过量值(ee),尤其是在低水含量反胶束中。当CHAPS浓度为8 mM时,AOT/CHAPS复配反胶束体系中上述反应48 h的产率可达34.2%,约为相同水含量下单一 AOT反胶束体系的两倍,产物的ee值由89.1%提高到98.2%。本文采用动态光散射、荧光发射光谱、傅里叶变换红外光谱等技术对反胶束的尺寸、增溶脂肪酶的构象及本体水的活性进行进一步表征。结果表明,CHAPS的加入增加了低水含量下AOT反胶束的尺寸,稳定了胶束中增溶脂肪酶的构象,降低了胶束中本体水的亲核性,以上因素共同促成上述脂肪酶催化酯合成反应的正向进行。本研究为反胶束体系中脂肪酶催化手性醇酸的制备提供了科学依据。2、甲基特丁基醚对AOT稳定的微乳液相行为及双连续微乳液中脂肪酶催化丁酸苏合香酯选择性水解反应的研究MTBE(甲基特丁基醚)是一种极性大的非质子型有机溶剂,在非水相酶催化领域表现出了独特的优势。为构建AOT稳定的MTBE基的双连续微乳液体系,本文通过T-γ鱼形相图法探究了 CHAPS对H2O/AOT/MTBE微乳液体系相行为的影响。结果表明,随着CHAPS质量分数的提高,微乳液体系的相反转温度持续降低,相应的BME体系中增溶脂肪酶的催化活力及其催化的丁酸苏合香酯选择性水解反应的产率不断增大。相较于异辛烷,相同组成下MTBE基BME体系中增溶脂肪酶的催化稳定性更好。本文选取了相反转温度适中的BME体系进一步探究表面活性剂含量和缓冲液pH对脂肪酶及其催化苏合香酯选择性水解反应的影响,结合微乳液微结构与脂肪酶构象探讨了相关的影响机制,优化后的MTBE基BME介质中18 h时的产物产率可达43%,对映体过量值接近100%。本研究为介质在脂肪酶促手性芳基醇/酸的高效生物制造领域中的推广应用提供了实验支撑。更多还原

【Abstract】 Lipase is an important biocatalyst for the stereoselective synthesis of chiral aromatic alcohols/acids.To achieve medium compatibility between hydrophilic lipase and its hydrophobic substrate,microemulsion is usually take as a medium for lipase-based catalytic reaction.There are many types of microemulsion.For different lipase-catalyzed reactions,a suitable type of microemulsion could be formulated by adjusting its composition,which is helpful for the efficient biopreparation of chiral aryl alcohols/acids in microemulsion.To the end,the present thesis focuses on the following two aspects:1.Effect of CHAPS on the selective synthesis of(S)-n-octyl ibuprofen ester catalyzed by lipase in AOT reverse micelleIn this work,the effect of biocompatible facial zwitterionic surfactant CHAPS(3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate)on the microstructure of AOT(sodium bis(2-ethylhexyl)sulfosuccinate)reversed micelle and the selective synthesis of(S)octyl ibuprofen ester catalyzed by lipase were investigated.The results demonstrate that CHAPS enhances both the yield and the enantiomeric excess value(ee)of the enzymatic reaction in AOT reversed micelle,especially at low water content.With 8 mM CHAPS in AOT reverse micelle,the yield of AOT/CHAPS mixed reversed micellar system in 48 h could reach 34.2%,which is approximately twice that without CHAPS,and the ee value of the product is increased from 89.1%to 98.2%.To understand the mechanism of the CHAPS effect,dynamic light scattering,fluorescence emission spectroscopy and Fourier transform infrared spectroscopy were used to characterize the size of reversed micelle,the conformation of solubilized lipase and the activity of bulk water.It reveals that the addition of CHAPS increases the size of AOT reverse micelles at low water content,stabilizes the conformation of lipase in micelles,and reduces the nucleophilicity of the bulk water in AOT reverse micelle.All the above factors provide a scientific support for the synthesis of chiral alcohols and acids catalyzed by lipase in reverse micelles.2.Construction of AOT-stabilized MTBE-based bicontinuous microemulsion(BME)for the selective hydrolysis of α-phenylethyl butyrate catalyzed by lipaseMethyl tert-butyl ether(MTBE),a polar aprotic organic solvent,demonstrates some advantages in the field of non-aqueous enzyme catalysis.To construct AOT-stabilized MTBEbased bicontinuous microemulsion,the influence of CHAPS on the phase behavior of the H2O/AOT/MTBE microemulsion system was investigated by T-γ fish phase diagram method.With the increase of CHAPS mass fraction in the surfactant,the phase inversion temperature of the microemulsion system is decreased,the catalytic activity of solubilized lipase and the yield of the selective enzymatic hydrolysis of α-phenylethyl butyrate is increased accordingly.The catalytic stability of solubilized lipase in the MTBE-based BME is higher than the isooctanebased BME with the same composition.For a selected MTBE-based BME with a proper phase inversion temperature,the effects of surfactant content and buffer pH on the solubilized lipase and the selective hydrolysis reaction is further explored,and the related influence mechanism is discussed together with the characterization of the microemulsion microstructure and lipase conformation.Under the optimized medium condition,a maximum product yield of 43%could be achieved in 18 h,and the ee value of the product is approaching 100%.This present study provides experimental support for the development of the efficient medium for the lipasecatalyzed synthesis of chiral aromatic alcohols/acids.更多还原