巯基准聚轮烷制备及其与胰凝乳蛋白酶和脲酶的相互作用

发布时间：2018-11-05 21:07

【摘要】：准聚轮烷是一种线形超分子组装体,在纳米器件、传感器、分子转换器、体内给药释放系统、基因释放载体以及组织工程支架材料等方面具有广泛的应用。本课题以聚乙二醇(PEG)/?-环糊精(CyD)准聚轮烷为载体,在PEG末端修饰巯基,制备一种氧化还原可控型的准聚轮烷水凝胶,测定胰凝乳蛋白酶和脲酶在水凝胶中的活性和结构的变化,探究此三元体系的相互作用,并对准聚轮烷的成核机理进行了初步的探索。通过酯化法和三步合成法制备了巯基聚乙二醇,通过FTIR、Raman、1H NMR表征发现在本实验室条件下,酯化法比较容易合成PEG?SH,产率70%以上。用?-CyD进行穿环反应制备巯基准聚轮烷,利用XRD对巯基准聚轮烷进行晶型分析,表明其属于六方晶系。为探索巯基准聚轮烷能否作为一种软固定化酶的材料,我们研究了胰凝乳蛋白酶在PEG?SH/?-CyD准聚轮烷体系中的活性和结构的变化。实验表明,在PEG?SH/?-CyD准聚轮烷体系中,胰凝乳蛋白酶的酶活提高了19%,稳定性也有明显提高。通过荧光光谱、同步荧光光谱、1H NMR、13C NMR、COSY、NOESY、圆二色谱的研究发现这主要是由于溶液中的胰凝乳蛋白酶与巯基准聚轮烷形成氢键、疏水键、范德华力等次级相互作用造成胰凝乳蛋白酶的结构发生轻微的变化,最终引起酶的催化效率提高。另外,巯基具有提高胰凝乳蛋白酶酶活的作用。以脲酶作为胰凝乳蛋白酶的对照酶,研究了脲酶/巯基准聚轮烷的相互作用。实验表明,巯基准聚轮烷对脲酶的活性和稳定性并没有影响。而在巯基浓度比较高的巯基乙酸(TGA)溶液中,脲酶迅速失活。考察脲酶的荧光光谱发现巯基准聚轮烷会降低酶的荧光强度,溶液中Trp、Tyr残基的微环境也会随着巯基准聚轮烷的加入而发生变化。此外,本课题还对准聚轮烷的相变成核机理进行了初步的探索。在0.1 g/mL?-CyD溶液中,准聚轮烷的形成速率会随着PEG浓度的增加而增加,并最终稳定在5?10-4 mg/mL?s左右。准聚轮烷的穿环及相变过程中,粒径不断增大,最终形成的准聚轮烷的粒径大概在1.5~3?m的范围内。由zeta电位的测试结果可知,在穿环过程中,一旦生成凝结核之后,凝结核迅速聚集,形成不稳定的颗粒体系,并发生相变。
[Abstract]:Quasi-polypropane is a linear supramolecular assembly, which has been widely used in nanodevices, sensors, molecular converters, drug delivery systems in vivo, gene release carriers and scaffold materials for tissue engineering. In this paper, a kind of redox controllable quasi polyether hydrogel was prepared by modifying sulfhydryl group at the end of PEG with polyethylene glycol (PEG) /? cyclodextrin (CyD) quasi polyether as carrier. The changes of activity and structure of chymotrypsin and urease in hydrogel were determined, the interaction of this ternary system was explored, and the nucleation mechanism of polyrotane was explored. Mercapto polyethylene glycol was prepared by esterification and three-step synthesis. FTIR,Raman,1H NMR characterization showed that the yield of PEG?SH, was more than 70% under the condition of esterification. Mercapto quasi polypropanes were prepared by the piercing reaction of?-CyD. The crystal structure of mercapto quasi-polypropanes was analyzed by XRD. The results show that they belong to hexagonal crystal system. In order to explore whether mercapto quasi-polypropane can be used as a soft immobilized enzyme, we have studied the changes of activity and structure of chymotrypsin in PEG?SH/?-CyD system. The results showed that the activity of chymotrypsin was increased by 19% and the stability of chymotrypsin was also improved in the PEG?SH/?-CyD system. Through fluorescence spectrum, synchronous fluorescence spectrum and 1H NMR,13C NMR,COSY,NOESY, circular dichroism, it was found that chymotrypsin in solution formed hydrogen bond and hydrophobic bond with mercapto quasipolypyridine, which was mainly due to the formation of hydrogen bond and hydrophobic bond between chymotrypsin and mercapto pseudopolymerane. Secondary interactions such as van der Waals lead to minor changes in the structure of chymotrypsin, resulting in an increase in the catalytic efficiency of chymotrypsin. In addition, sulfhydryl group can increase the activity of chymotrypsin. The interaction between urease and mercapto quasi-polypropanes was studied by using urease as a control enzyme of chymotrypsin. The results showed that mercapto quasi-polybutane had no effect on the activity and stability of urease. However, urease was rapidly deactivated in (TGA) solution with higher concentration of mercaptoacetic acid. The fluorescence spectra of urease showed that mercapto quasi polybutane could decrease the fluorescence intensity of the enzyme, and the microenvironment of Trp,Tyr residues in the solution would change with the addition of mercapto quasi polybutane. In addition, the mechanism of phase transition nucleation of polyrotane was also explored. In 0. 1 g/mL?-CyD solution, the formation rate of quasi-polybutane increased with the increase of PEG concentration, and finally stabilized at about 5 ~ 10-4 mg/mL?s. In the process of ring penetration and phase transition, the particle size of quasi-polybutane is increasing, and the particle size of the resulting quasi-polybutane is in the range of 1. 5 ~ 3? M. From the results of zeta potential measurement, it can be seen that, once the condensation nucleus is formed, the condensate nucleus accumulates rapidly, forming an unstable particle system and a phase transition occurs during the annular penetration.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O641.3

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