【作者】 李明；

【导师】 李在均；

【作者基本信息】 江南大学 ， 应用化学， 2014， 博士

【摘要】 随着绿色化学在全球的倡导及消费者对天然香料日益强烈的追求，生物仿生合成天然香料成为香料工业最具发展潜力的途径之一。离子液体是一种环境友好的绿色溶剂，可替代易挥发性有机溶剂应用于非水生物催化。然而，迄今人们对离子液体对酶行为的影响机制和规律性了解甚少。为此，本论文开展了新型对称烷基咪唑型离子液体的设计、合成、性质及作为天然香料仿生合成介质的研究。设计合成出十种阳离子是1,3-二烷基咪唑[D(R)IM]+而阴离子为[PF6]-和[OAc]-的新型对称烷基咪唑型离子液体。其中，四种对称烷基咪唑醋酸盐离子液体未见文献报道。研究发现，阳离子咪唑环1、3位N上取代基的碳链长短与同分异构之间的构型差异都会显著影响离子液体的性质。当阳离子相同时，[OAc]-类离子液体的密度、热稳定性、粘度、极性及表面张力都要低于相应的[PF6]-与[NTf2]-离子液体。考察离子液体的提取、富集和定香性能，研究离子液体与香料分子间的相互作用。离子液体提取芳樟油表明，具有较高极性与较低粘度的离子液体对芳樟油的提取率较高。SEM结果证实离子液体可以有效破坏植物组织细胞，从而有助于精油的释放。离子液体顶空单滴微萃取酯类香料结果显示，离子液体对酯类化合物的富集倍数既受其憎水性、极性和粘度的影响，还与分析物本身的结构及性质密切相关。良好的憎水性、较低的极性有利于富集，而高粘度则相反。离子液体对芳香族酯类的富集倍数最高，萜类酯其次，饱和脂肪族酯相对最低。离子液体对香料的定香性能研究发现，具有较高极性的离子液体与极性较大的香料如醇类化合物之间的作用力较强，而低极性的离子液体与弱极性的香料如酮类、酯类、萜类化合物之间的作用力较强，高粘度有助于离子液体对香料的定香作用。分别以脂肪酶催化(-)-薄荷醇与乙酸酐酯化合成(-)-乙酸薄荷酯及脂肪酶催化拆分(±)-薄荷醇为模型反应，考察不同离子液体介质对酶行为的影响。结果发现酶在离子液体中的活性、立体选择性和稳定性显著优于有机溶剂正己烷，(-)-薄荷醇转化率高达97.4%，(±)-薄荷醇拆分反应转化率与e.e.值分别可达48.1%与98.1%。荧光光谱和圆二色谱研究结果表明，酶在离子液体中有较大的酶蛋白分子裸露程度和良好的二级结构稳定性。以上事实表明离子液体的阳离子尺寸及空间构型是影响酶行为的重要因素。采用新型功能导电聚合物共价键合脂肪酶固定于铜网电极上，然后涂覆离子液体构建新型生物反应器。以催化拆分(R,S)-1-苯乙醇为模型反应，考察酶的活性、稳定性和重复利用性。结果表明，所制备的生物反应器具有良好的酶催化活性与立体选择性，在最佳反应条件下，最初反应速率、反应转化率及e.e.值分别为1.76Umg-1、47.4%和99.0%。固定化酶的半衰期是游离酶的5.1倍，生物反应器重复使用10次后酶活性没有明显减少。共价键合方式固定酶及离子液体良好的亲生物性使生物传感器在酶活性和稳定性方面得到较大提高。更多还原

【Abstract】 With the development of new green chemistrymethodologiesandthe dramaticallyincreasing demand of consumers fornatural flavours, biocatalysis has become one of the mostpromising approaches for the synthesis of natural flavours inthe fragrance and flavourindustry. Being valuable environmentally benign media, ionic liquids (ILs) have recentlyappeared as clean alternatives to volatile organic solvents for a wide variety of non-aqueousenzymatic reactions. However, so far, the researches on the influence of ionic liquids onenzyme are preliminary ones. In particular, the effects and mechanism of ionic liquids onenzyme are poorly understood. In the present work, the design, synthesis and characterizationof novel symmetrical alkylimidazolium ILs were studied, and the enzymatic syntheses ofnatural flavours were assayed with different ILs as media.Ten novel symmetrical alkylimidazolium ILs were designed and synthesized. The cationswere1,3-dialkylimidazolium [D(R)IM]+, while the anions mainly consisted of PF6-and OAc-.Among these ILs, four acetate ILs had not been reported before. It was found that the alkylchain length at the1-and3-position of nitrogen of the imidazole cation, as well as the spatialconfiguration of isomers, would significantly affect the properties of IL. When the cationremained the same, the density, thermal stability, viscosity, polarity and surface tension of theacetate ILs were lower than those of hexafluorophosphate ILs and bis-(trifluoromethyl-sulfonyl) imide ILs.The extraction performance, the enrichment performance and the lasting fragranceperformance of novel ILs were investigated. Furthermore, interactions between ILs andflavour molecules were explored. The ILs were employed as the solvent for extraction of theessential oil from Cinnamomum camphora presl leaves. It was found that the IL with higherpolarity and lower viscosity offered a better capacity for the extraction. SEM resultsdemonstrated that IL treatment disintegrated the plant materials efficiently thus increasing therelease of essential oil. The ILs were chosen as the extractant based headspace single-dropmicro-extraction for esters flavors. The results showed that the extraction efficiency dependnot only on the viscosity, hydrophobicity and polarity of the IL, but also on the structure andproperties of the analyte. More specifically, good hydrophobicity and low polarity of ILs helpthe enrichment of ester compounds, whereas high viscosity shows opposite effects. Besides,the enrichment factors of aromatic esters were highest, followed by terpene esters andsaturatedaliphatic esters. The ILs were employed as the fixative of the fragrant compoundswith different functional groups. It was observed that relative high-polarity IL had a stronginteraction with polar flavour molecule such as alcohols. In constrast, relative weak-polarityIL had a strong interaction with weak polar flavour molecule such as ketones, esters andterpenes. Additionally, IL with high viscosity offered good lasting fragrance efficiency.The enzymaticsynthesis of (-)-menthyl acetate by esterificationfrom (-)-menthol andacetic anhydride catalyzed by Pseudomonas cepacialipase and the enzymatic kineticresolution of (±)-menthol over Candida antarctica lipaseby using propionic anhydride as theacyl donor in the acylation reaction were chosen as model reaction to investigate the properties of lipase in different ILs, respectively. The results showed that the reactivity,selectivity and stability of the lipase in IL medium were obviously better than those in organicsolvent hexane. Under optimal reaction conditions, the conversion of (-)-menthol was up to97.4%, the conversion of (±)-menthol and the enantiomeric excess of (-)-menthyl propionatewas48.1%and98.1%, respectively. Moreover, fluorescence spectroscopy and circulardichroism results showed that the lipase in IL had slightly big exposure level of amino acidresidues and excellent secondary structure stability. The fact above also revealed that not onlydoes the alkyl chain length of the imidazole cation affect the efficiency of enzyme in estersynthesis but also the spatial configuration of the IL plays an important role in overall enzymeactivity.A new functional conducting polymer was developed to immobilize lipase on copper netand the ionic liquid was finally coated on the surface of the modified electrode to fabricate anovel bioreactor. The resolution of (R,S)-1-phenylethanol was selected as a model reaction toinvestigate the activity, stability and reuse of the enzyme immobilized on the bioreactor. Theresults showed that the enzyme immobilized on the bioreactor exhibited high catalytic activityand selectivity. Under optimal conditions, initial reaction rate, the conversion of(R,S)-1-phenylethanol and enantiomeric excess of (R)-1-phenylethyl acetate was1.76Umg-1,47.4%and99%, respectively. Also, the lipase immobilized on the bioreactor was recycled10times without substantial diminution in activity and its half life time was5.1-fold that of thenative lipase in hexane. The functional conducting polymer combined with the ionic liquid forthe enzyme immoblization provided good enzyme activity and stability.更多还原