DESs中酶催化制备糖苷及苷元类活性物质研究

发布时间：2018-06-27 12:06

本文选题：低共熔溶剂 + 非水相酶催化　；参考：《山西大学》2017年硕士论文

【摘要】：糖苷及苷元类化合物广泛分布于自然界中,具有抗肿瘤、抗氧化、抗疲劳以及防治心脑血管疾病等显著药理活性,在药物化学领域是一类重要的药用资源化合物。然而,由于植物资源的匮乏以及自身含量的低微,糖苷及苷元类化合物的需求不得不依靠于人工制备,如有机合成、组织培养以及酶催化生物转化等。众所周知,化学合成法及组织悬浮培养法均存在或多或少的缺陷(周期长且产率低等),制约了其进一步发展应用。不可置否,酶催化生物转化过程具有反应条件温和、高效简便且环境友好的显著优势,是糖苷及苷元类活性化合物制备的理想途径,极具发展潜力。酶催化反应过程大多选择在传统缓冲液中进行,但受制于某些反应类型特性以及底物溶解性的差异,水相并不是适宜的反应溶剂体系。鉴于上述情况,非水相酶催化应用逐步发展,可以促进传统水相中难以进行或效率极低的催化反应过程的发生,极大顺应了酶工程学领域的应用要求。低共熔溶剂(Deep Eutectic Solvents,DESs)是一类新型绿色溶剂,自2003年问世以来便凭借其独特的理化性质及优良特性而广受关注,并在非水相酶催化反应的应用中显示出了独特的优越性。基于此,本研究选用DESs含水溶液为酶催化反应溶剂,首次以β-D-葡萄糖苷酶为研究对象,初步探讨了不同DESs介质对于糖苷酶催化特性的作用规律及影响机制;选用不同催化机制对应的模型反应,通过最佳工艺的优化及放大制备研究确定了该反应体系在酶催化制备糖苷及苷元类活性物质的可行性及先进性。在本论文中,首先,我们合成了16种氯化胆碱型DESs,考察了苦杏仁来源β-D-葡萄糖苷酶在上述DESs介质中的酶催化活性、稳定性(热稳定性及存储稳定性),并筛选出了整体表现较优的候选DESs。在上述候选DESs中,进一步测定了酶的最适反应温度、pH值及米氏常数Km等酶学性质参数。实验结果表明,DESs介质对于苦杏仁来源β-D-葡萄糖苷酶催化特性具有显著影响,酶催化活性略有降低,但稳定性却显著提升,不同DESs类型的影响主要取决于氢键供体种类以及组成成分的摩尔比例。在维持酶的相对活力方面,多元醇类DESs表现明显优于酰胺类;当氢键受体与供体摩尔比例为1:1时,酶的相对活力为不同比例中最低值,而组成为其它比例(2:1,1:1,1:3)时则表现相当。低浓度DESs体系中,酶的最适反应温度及pH值较缓冲液中无明显改变,而酶促动力学参数Km以及Vmax均存在一定程度下降,表明底物与酶分子的诱导契合作用增强,但底物的传质扩散却受到抑制。第二,将所选DESs介质ChCl/G(1:2)作为反应体系,研究了酶催化糖基化合成红景天苷的可行性。利用单因素试验与响应面优化法,确定了壳聚糖固定化的β-D-葡萄糖苷酶催化糖基化合成红景天苷反应最佳工艺条件:DESs浓度为80 vol%,反应温度50°C,体系pH值为5.8,反应时间100 h,酶添加量为45 U/m L,底物酪醇/葡萄糖摩尔比例为10;在此条件下,底物最大转化率可达31.6%,经大孔树脂层析初步提纯,红景天苷粗产品纯度可达70%以上。此外,所采用的固定化酶也可循环利用,具有较高的操作稳定性,保障了反应的可行性以及可持续性。第三,评价了DESs介质中酶催化水解制备大豆苷元的可行性与适用性。经单因素试验与响应面优化法确定最佳工艺条件为:DESs浓度为30 vol%,反应时间为100min,反应温度为55°C,pH值为5.4,酶用量为1.0 U/mL,底物浓度为300μg/m L;在此条件下,底物最大转化率高达97.53%。按上述工艺条件进行放大制备,产物大豆苷元在反应过程中可以不断析出,简单过滤即可实现产物分离,而体系可继续投料直接进行下一步反应,可重复利用5次以上。此外,对于酶催化大豆苷水解反应而言,我们发现DESs介质对于底物大豆苷具有优良的溶解性能,在大规模生产中具有更高的适用价值。第四,系统考察了不同DESs体系对于大豆苷及大豆苷元的溶解特性规律。结果显示,在室温下,大豆苷及大豆苷元分别在ChCl/EG(1:2,50 vol%)和ChCl/U(1:2,50vol%)体系中溶解效果最优,各自平衡溶解度分别达475.4μg/m L和993.6μg/m L。溶解过程中,我们发现DESs浓度对于溶质溶解度影响较为显著,但二者呈现出不同规律;在测定溶质共存溶解度时,研究表明大豆苷在一定程度上可以促进大豆苷元的溶解,推测当联合使用时可以提高其体内的溶解吸收。经综合筛选,最终选用ChCl/U(1:2)/ChCl/EG(1:2)(v/v,1:7;80,100 vol%)体系作为混合溶剂,采用不同热力学方程进行关联拟合,其中Apelblat方程关联度最优,相关系数R2均达到0.999以上。最后,我们利用紫外吸收以及荧光发射光谱技术,初步研究了不同种类DESs介质对于β-D-葡萄糖苷酶结构的影响规律,从蛋白分子构象角度分析了DESs体系影响酶催化特性的基础作用机理。在DESs体系中,酶的紫外以及荧光光谱均发生了最大波长位移以及强度的改变,主要原因可能是由于溶剂效应引起的酶蛋白分子中氨基酸侧链的暴露,从而引起构象改变所致。研究表明,DESs介质中酶蛋白光谱的变化与其催化活性及稳定性呈现一定关联性,DESs介质对于酶催化特性的影响主要是由于酶分子的蛋白结构发生了改变。总而言之,本论文首次研究了β-D-葡萄糖苷酶在DESs介质中的作用规律及可行性,成功构建了酶催化制备糖苷以及苷元类化合物的新型绿色体系,具有高效简便、环境友好等显著优势,展现出一定的实用价值,同时也可为此领域的相关学术研究提供必不可少的参考思路和理论依据。
[Abstract]:Glycosides and glycosides are widely distributed in nature, with significant pharmacological activities such as anti-tumor, antioxidation, anti fatigue and prevention and treatment of cardiovascular and cerebrovascular diseases. It is a kind of important medicinal resource compound in the field of drug chemistry. However, due to the lack of plant resources and the low content of its own, the need for glycosides and glucoside compounds We have to rely on artificial preparation, such as organic synthesis, tissue culture and enzyme catalyzed biotransformation. It is well known that chemical synthesis and tissue suspension culture have more or less defects (long period and low yield), which restricts its further development. The remarkable advantage of high efficiency, simple and environmentally friendly is an ideal way for the preparation of glycosides and active compounds of glycosides. The catalytic reaction process is mostly selected in the traditional buffer solution, but it is subject to some types of reaction characteristics and the difference of substrate solubility. The aqueous phase is not a suitable reaction solvent system. In this case, the non-aqueous enzymatic catalytic application is progressively developed, which can promote the development of the catalytic reaction process in the traditional water phase which is difficult to carry out or with very low efficiency. It has greatly complied with the application requirements of the field of enzyme engineering. Deep Eutectic Solvents (DESs) is a new type of green solvent, which has been unique since its advent in 2003. In this study, DESs aqueous solution was used as a solvent for enzymatic reaction. The effect of different DESs media on the catalytic properties of glucosidase was preliminarily studied in this study. In this paper, 16 kinds of choline chloride type DESs were combined into 16 kinds of choline chloride type. In this paper, we studied the bitter almond. The enzyme catalytic activity of the source beta -D- glucosidase in the DESs medium, stability (thermal stability and storage stability), and screening the candidate DESs. with better overall performance in the above candidate DESs, and further measuring the enzyme's optimum reaction temperature, pH value and Michaelis constant Km and other enzymology properties. The experimental results show that DESs medium is used. The catalytic properties of amygdala derived beta -D- glucosidase had a significant influence. The catalytic activity of the enzyme decreased slightly, but the stability was significantly improved. The effects of different DESs types mainly depend on the type of hydrogen bond and the molar ratio of the components. The DESs performance of the polyols is obviously superior to the amides in maintaining the relative activity of the enzyme. When the ratio of the bond receptor to the donor molar ratio is 1:1, the relative activity of the enzyme is the lowest in the different proportion, while the composition is the same as the other proportion (2:1,1:1,1:3). In the low concentration DESs system, the optimum reaction temperature and the pH value of the enzyme are not significantly changed in the buffer solution, but the enzyme kinetic parameters Km and Vmax are all decreased to a certain extent, indicating that the enzyme kinetic parameters, Km and Vmax, have been reduced to a certain extent. The interaction between the substrate and the enzyme molecule is enhanced, but the mass transfer and diffusion of the substrate is inhibited. Second, the feasibility of enzyme catalyzed glycosylation of salidroside was studied by using the selected DESs medium ChCl/G (1:2) as a reaction system. The immobilized beta -D- glucosidase catalysis of chitosan was determined by single factor test and response surface optimization. The optimum process conditions for glycosylation of salidroside are: DESs concentration is 80 vol%, reaction temperature is 50 degree C, pH value is 5.8, reaction time is 100 h, enzyme addition is 45 U/m L, and the molar ratio of substrate Tyrol / glucose is 10. Under this condition, the maximum conversion rate of substrate can reach 31.6%, and the crude product of salidroside is pure and pure by macroporous resin chromatography In addition, the immobilized enzyme can also be recycled, with high operating stability, the feasibility and sustainability of the reaction. Third, the feasibility and applicability of the enzyme catalyzed hydrolysis of Daidzein in DESs medium was evaluated. The optimum process conditions were determined by single factor test and response surface optimization. The DESs concentration is 30 vol%, the reaction time is 100min, the reaction temperature is 55 degree C, the pH value is 5.4, the enzyme dosage is 1 U/mL, the substrate concentration is 300 mu g/m L. Under this condition, the maximum conversion rate of the substrate is up to 97.53%. according to the above process conditions, and the product daidzein can be continuously precipitated during the reaction process, and the product can be realized by simple filtration. In addition, we found that the DESs medium has excellent solubility in the daidzein, and has a higher suitable value in the large-scale production. Fourth, the system has been examined by different DESs systems for the enzyme catalyzed daidzein hydrolysis. The dissolution characteristics of daidzein and daidzein showed that, at room temperature, daidzein and daidzein were best dissolved in ChCl/EG (1:2,50 vol%) and ChCl/U (1:2,50vol%) system respectively. The equilibrium solubility of daidzein and ChCl/U (1:2,50vol%) was 475.4 u g/m L and 993.6 micron g/m L. dissolution process respectively. We found that the concentration of DESs was affected by the solubility of the solute. More significant, but the two showed different rules. In determining the solubility of solute, the study showed that daidzein could promote the dissolution of daidzein to a certain extent. It is presumed that the dissolution and absorption of daidzein can be improved when combined. After comprehensive screening, the ChCl/ U (1:2) /ChCl/EG (1:2) (v/v, 1:7; 80100 vol%) system is selected as the final selection. In the mixed solvent, the correlation of the Apelblat equation was optimal and the correlation coefficient R2 reached 0.999. Finally, we studied the effect of different kinds of DESs media on the structure of beta -D- glucosidase by UV absorption and fluorescence emission spectroscopy. The basic mechanism of the effect of DESs system on the catalytic properties of enzymes is analyzed. In the DESs system, the maximum wavelength shift and the intensity change of the enzyme in the UV and fluorescence spectra are caused by the exposure of the amino acid side chain in the enzyme protein molecules caused by the solvent effect, and the result of the conformation changes. It is clear that the changes of the enzyme protein spectrum in the DESs medium are related to its catalytic activity and stability. The effect of DESs medium on the enzyme catalytic properties is mainly due to the changes in the protein structure of the enzyme molecules. In a word, the role and feasibility of the beta -D- glucosidase in the DESs medium is first studied in this paper. A new green system with enzyme catalyzed preparation of glycosides and glucoside compounds has been built. It has significant advantages, such as high efficiency, simplicity, environment friendly and so on. It shows a certain practical value. At the same time, it can also provide a necessary reference and theoretical basis for the related academic research in this field.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914

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